CA3142883A1

CA3142883A1 - Engineered casx systems

Info

Publication number: CA3142883A1
Application number: CA3142883A
Authority: CA
Inventors: Benjamin OAKES; Sean Higgins; Hannah SPINNER; Sarah DENNY; Brett T. STAAHL; Kian TAYLOR; Katherine BANEY; Isabel COLIN; Maroof ADIL
Original assignee: Scribe Therapeutics Inc
Current assignee: Scribe Therapeutics Inc
Priority date: 2019-06-07
Filing date: 2020-06-05
Publication date: 2020-12-10
Also published as: US20220220508A1; EP3980533A1; GB2600274A; PE20220256A1; CL2021003233A1; TW202113074A; KR20220032050A; WO2020247882A1; AU2020289591A1; US20230124880A1; US20220081681A1; JP2022534809A; IL288738A; MX2021015058A; SG11202113253SA; US11560555B2; BR112021024288A2; CN114375334A

Abstract

Provided herein are engineered CasX systems and components thereof, including variant CasX proteins and variant guide nucleic acids (gNAs). The variant CasX proteins and variant gNAs of the disclosure display at least one improved characteristic when compared to a reference CasX protein or reference gNA of the disclosure. In some instances, the variants have one or more improved CasX ribonucleoprotein complex functions. Also provided are methods of making and using said variants.

Description

DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.

NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME

NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME:
NOTE POUR LE TOME / VOLUME NOTE:

2 PCT/US2020/036505 ENGINEERED CASX SYSTEMS
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional patent application numbers 62,858,750, filed on June 7, 2019, 62/944,892, filed on December 6, 2019 and 63/030,838, filed on May 27, 2020, the contents of each of which are incorporated herein by reference in their entireties.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
[0002] This application contains a Sequence listing which has been submitted in ASCII format via EFS-WEB and is hereby incorporated by reference in its entirety. Said ASCII copy, created on June 5,2020 is named SCRB 01103W0 SeqList 25 and is 3.63 MB in size.
BACKGROUND

[0003] The CRISPR-Cas systems confer bacteria and archaea with acquired immunity against phage and viruses. Intensive research over the past decade has uncovered the biochemistry of these systems. CRISPR-Cas systems consist of Cas proteins, which are involved in acquisition, targeting and cleavage of foreign DNA or RNA, and a CRISPR array, which includes direct repeats flanking short spacer sequences that guide Cas proteins to their targets. Class 2 CRISPR-Cas are streamlined versions in which a single Cas protein bound to RNA is responsible for binding to and cleavage of a targeted sequence. The programmable nature of these minimal systems has facilitated their use as a versatile technology that is revolutionizing the field of genome manipulation.

[0004] To date, only a few Class 2 CRISPR/Cas systems have been discovered that have been widely used. Thus, there is a need in the art for additional Class 2 CRISPR/Cas systems (e.g., Cas protein plus guide RNA combinations) that have been optimized and/or offer improvements over earlier generation systems for utilization in a variety of therapeutic, diagnostic, and research applications.
SUMMARY

[0005] In some aspects, the present disclosure provides variants of a reference CasX nuclease protein, wherein the CasX variant is capable of forming a complex with a guide nucleic acid (NA), and wherein the complex can bind a target DNA, wherein the target DNA
comprises non-target strand and a target strand, and wherein the CasX variant comprises at least one modification relative to a domain of the reference CasX and exhibits one or more improved characteristics as compared to the reference CasX protein. The domains of the reference CasX
protein include: (a) a non-target strand binding (NTSB) domain that binds to the non-target strand of DNA, wherein the NTSB domain comprises a four-stranded beta sheet;
(b) a target strand loading (TSL) domain that places the target DNA in a cleavage site of the CasX variant, the TSL domain comprising three positively charged amino acids, wherein the three positively charged amino acids bind to the target strand of DNA, (c) a helical I domain that interacts with both the target DNA and a spacer region of a guide NA, wherein the helical I
domain comprises one or more alpha helices; (d) a helical II domain that interacts with both the target DNA and a scaffold stem of the guide NA; (e) an oligonucleotide binding domain (OBD) that binds a triplex region of the guide NA; and (f) a RuvC DNA cleavage domain.

[0006] In some aspects, the present disclosure provides variants of a reference guide nucleic acid (gNA) capable of binding a CasX protein, wherein the reference guide nucleic acid comprises at least one modification in a region compared to the reference guide nucleic acid sequence, and the variant exhibits one or more improved characteristics compared to the reference guide RNA. The regions of the scaffold of the gNA include: (a) an extended stem loop; (b) a scaffold stem loop; (c) a triplex; and (d) pseudoknot. In some cases, the scaffold stem of the variant gNA further comprises a bubble. In other cases, the scaffold of the variant gNA
further comprises a triplex loop region. In other cases, the scaffold of the variant gNA further comprises a 5' unstructured region.

[0007] In some aspects, the present disclosure provides gene editing pairs comprising the CasX proteins and gNAs of any of the embodiments described herein.

[0008] In some aspects, the present disclosure provides polynucleotides and vectors encoding the CasX proteins, gNAs and gene editing pairs described herein. In some embodiments, the vectors are viral vectors such as an Adeno-Associated Viral (AAV) vector or a lentiviral vector.
In other embodiments, the vectors are non-viral particles such as virus-like particles or nanoparticles.

[0009] In some aspects, the present disclosure provides cells comprising the polynucleotides, vectors, CasX proteins, gNAs and gene editing pairs described herein. In other aspects, the present disclosure provides cells comprising target DNA edited by the methods of editing embodiments described herein.

[0010] In some aspects, the present disclosure provides kits comprising the polynucleotides, vectors, CasX proteins, gNAs and gene editing pairs described herein.

[0011] In some aspects, the present disclosure provides methods of editing a target DNA, comprising contacting the target DNA with one or more of the gene editing pairs described herein, wherein the contacting results in editing of the target DNA.

[0012] In other aspects, the disclosure provides methods of treatment of a subject in need thereof, comprising administration of the gene editing pairs or vectors comprising or encoding the gene editing pairs of any of the embodiments described herein.

[0013] In another aspect, provided herein are gene editing pairs, compositions comprising gene editing pairs, or vectors comprising or encoding gene editing pairs, for use as a medicament.

[0014] In another aspect, provided herein are gene editing pairs, compositions comprising gene editing pairs, or vectors comprising or encoding gene editing pairs, for use in a method of treatment, wherein the method comprises editing or modifying a target DNA;
optionally wherein the editing occurs in a subject having a mutation in an allele of a gene wherein the mutation causes a disease or disorder in the subject, preferably wherein the editing changes the mutation to a wild type allele of the gene or knocks down or knocks out an allele of a gene causing a disease or disorder in the subject.
BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0016] FIG. 1 is a diagram showing an exemplary method of making CasX protein and guide RNA variants of the disclosure using Deep Mutational Evolution (DME). In some exemplary embodiments, DME builds and tests nearly every possible mutation, insertion and deletion in a biomolecule and combinations/multiples thereof, and provides a near comprehensive and unbiased assessment of the fitness landscape of a biomolecule and paths in sequence space towards desired outcomes. As described herein, DME can be applied to both CasX
protein and guide RNA.

[0017] FIG. 2 is a diagram and an example fluorescence activated cell sorting (FACS) plot illustrating an exemplary method for assaying the effectiveness of a reference CasX protein or single guide RNA (sgRNA), or variants thereof A reporter (e.g. GFP reporter) coupled to a gRNA target sequence, complementary to the gRNA spacer, is integrated into a reporter cell line. Cells are transformed or transfected with a CasX protein and/or sgNA
variant, with the spacer motif of the sgRNA complementary to and targeting the gRNA target sequence of the reporter. Ability of the CasX:sgRNA ribonucleoprotein complex to cleave the target sequence is assayed by FACS. Cells that lose reporter expression indicate occurrence of CasX:sgRNA
ribonucleoprotein complex-mediated cleavage and indel formation.

[0018] FIG. 3A and FIG. 3B are heat maps showing the results of an exemplary DME
mutagenesis of the reference sgRNA encoded by SEQ ID NO: 5, as described in Example 3.
FIG. 3A shows the effect of single base pair (single base) substitutions, double base pair (double base) substitutions, single base pair insertions, single base pair deletions, and a single base pair deletion plus at single base pair substitution at each position of the reference sgRNA shown at top. FIG. 3B shows the effect of double base pair insertions and a single base pair insertion plus a single base pair substitution at each position of the improved reference sgRNA. The reference sgRNA sequence of SEQ ID NO: 5 is shown at the top of FIG. 3A and bottom of FIG. 3B. In FIG. 3A and FIG. B, Log2 fold enrichment of the variant in the DME library relative to the reference sgRNA following selection is indicated in grayscale. Enrichment is a proxy for activity, where greater enrichment is a more active molecule. The results show regions of the reference sgRNA that should not be mutated and key regions that are targeted for mutagenesis.

[0019] FIG. 4A shows the results of exemplary DME experiments using a reference sgRNA, as described in Example 3. The improved reference sgNA (an sgRNA) with a sequence of SEQ
ID NO: 5 is shown at top, and Log2 fold enrichment of the variant in the DME
library relative to the reference sgRNA following selection is indicated in grayscale. Enrichment is a proxy for activity, where greater enrichment is a more active molecule. The heat map shows an exemplary DME experiment showing four replicates of a library where every base pair in the reference sgRNA has been substituted with every possible alternative base pair.

[0020] FIG. 4B is a series of 8 plots that compare biological replicates of different DME
libraries. The Log2 fold enrichment of individual variants relative to the reference sgRNA
sequence for pairs of DME replicates are plotted against each other. Shown are plots for single deletion, single insertion and single substitution DME experiments, as well as wild type controls, and the plots indicate that there is a good amount of agreement for each replicate.

[0021] FIG. 4C is a heat map of an exemplary DME experiment showing four replicates of a library where every location in the reference sgRNA has undergone a single base pair insertion.
The DME experiment used a reference sgRNA of SEQ ID NO: 5 (at top), and was performed as described in Example 3. Log2 fold enrichment of the variant in the DME library relative to the reference sgRNA following selection is indicated in grayscale.

[0022] FIGS. 5A-5E are a series of plots showing that sgNA variants can improve gene editing by greater than two fold in an EGFP disruption assay, as described in Examples 2 and 3. Editing was measured by indel formation and GFP disruption in HEK293 cells carrying a GFP reporter.
FIG. 5A shows the fold change in editing efficiency of a CasX sgRNA reference of SEQ ID NO:
4 and a variant of the reference which has a sequence of SEQ ID NO: 5, across 10 targets. When averaged across 10 targets, the editing efficiency of sgRNA SEQ ID NO: 5 improved 176%
compared to SEQ ID NO: 4. FIG. 5B shows that further improvement of the sgRNA
scaffold of SEQ ID NO: 5 is possible by swapping the extended stem loop sequence for additional sequences to generate the scaffolds whose sequences are shown in Table 2. Fold change in editing efficiency is shown on the Y-axis. FIG. 5C is a plot showing the fold improvement of sgNA variants (including a variant with SEQ ID NO: 17) generated by DME
mutations normalized to SEQ ID NO: 5 as the CasX reference sgRNA. FIG. 5D is a plot showing the fold improvement of sgNA variants of sequences listed in Table 2, which were generated by appending ribozyme sequences to the reference sgRNA sequence, normalized to SEQ ID NO: 5 as the CasX reference sgRNA. FIG. 5E is a plot showing the fold improvement normalized to the SEQ ID NO: 5 reference sgRNA of variants created by both combining (stacking) scaffold stem mutations showing improved cleavage, DME mutations showing improved cleavage, and using ribozyme appendages showing improved cleavage. The resulting sgNA
variants yield 2 fold or greater improvement in cleavage compared to SEQ ID NO: 5 in this assay. EGFP editing assays were performed with spacer target sequences of E6 and E7.

[0023] FIG. 6 shows a Hepatitis Delta Virus (HDV) genomic ribozyme used in exemplary gNA variants (SEQ ID NOs: 18-22).

[0024] FIGS. 7A-7I are a series of heat maps showing the effect of single amino acid substitutions, single amino acid insertions, and deletions at each amino acid position in a reference CasX protein of SEQ ID NO: 2, as described in Example 4. Data were generated by a DME assay run at 37 C. The Y-axis shows each possible substitution or insertion (from top to bottom: R, H, K, D, E, S, T, N, Q, C, G, P, A, I, L, M, F, W, Y or V; boxes indicate the amino acid identity of the reference protein), the X-axis shows the amino acid position in the reference CasX protein. Log2 fold enrichment of the CasX variant protein relative to the reference CasX
protein of SEQ ID NO: 2 in a DME library following enrichment is indicated. As used herein, "enrichment" is a proxy for activity, where greater enrichment is a more active molecule. (*)s indicate active sites. FIGS. 7A-7D show the effect of single amino acid substitutions. FIGS. 7E-7H show the effect of single amino acid insertions. FIG. 71 shows the effect of single amino acid deletions.

[0025] FIGS. 8A-8C are a series of heat maps showing the effect of single amino acid substitutions, single amino acid insertions and deletions at each amino acid position in a reference CasX protein of SEQ ID NO: 2, as described in Example 4. Data were generated by a DME assay run at 45 C. FIG. 8A shows the effect of single amino acid substitutions. FIG. 8B
shows the effect of single amino acid insertions. FIG. 8C shows the effect of single amino acid deletions. For all of FIGS. 8A- 8C, The Y-axis shows each possible substitution or insertion (from top to bottom: R, H, K, D, E, S, T, N, Q, C, G, P, A, I, L, M, F, W, Y
or V; boxes indicate the amino acid identity of the reference protein), the X-axis shows the amino acid position in the reference CasX protein. Log2 fold enrichment of the CasX variant protein relative to the reference CasX protein of SEQ ID NO: 2 in a DME library following enrichment is indicated in grayscale, where greater enrichment is a more active molecule. (*)s indicate active sites.
Running this assay at 45 C enriches for different variants than running the same assay at 37 C
(see FIGS. 7A-7I), thereby indicating which amino acid residues and changes are important for thermostability and folding.

[0026] FIG. 9 shows a survey of the comprehensive mutational landscape of all single mutations of a reference CasX protein of SEQ ID NO: 2. On the Y-axis, fold enrichment of CasX variants relative to the reference CasX protein for single substitutions (top), single insertions (middle) or single deletions (bottom). On the X-axis, amino acid position in the reference CasX protein. Key regions that yield improved CasX variants are the initial helix region and regions in the RuvC domain bordering the target strand loading (TLS) domain, as well as others.

[0027] FIG. 10 is a plot showing that the evaluated CasX variant proteins improved editing greater than three-fold relative to a reference CasX protein in the EGFP
disruption assay, as described in Example 5. CasX proteins were tested for their ability to cleave an EGFP reporter at 2 different target sites in human HEK293 cells, and the normalized improvement in genome editing at these sites over the basic reference CasX protein of SEQ ID NO: 2 is shown. Variants, from left to right (indicated by the amino acid substitution, insertion or deletion at the given residue number) are: Y789T, [P793], Y789D, T725, I546V, E552A, A636D, F5365, A708K, Y797L, L792G, A739V, G791M, A3661, A788W, K390R, A7515, E385A, AP696, AM773, G695H, AA5793, AA5795, C477R, C477K, C479A, C479L, 155F, K210R, C2335, D231N, Q338E, Q338R, L379R, K390R, L481Q, F4955, D600N, T886K, A739V, K460N, I199F, G492P, T153I, R591I, AA5795, AA5796, AL889, E121D, 5270W, E712Q, K942Q, E552K, K25Q, N47D, AT696, L685I, N880D, Q102R, M734K, A7245, T704K, P224K, K25R, M29E, H152D, 5219R, E475K, G226R, A377K, E480K, K416E, H164R, K767R, I7F, M29R, H435R, E385Q, E385K, I279F, D4895, D732N, A739T, W885R, E53K, A238T, P283Q, E292K, Q628E, R388Q, G791M, L792K, L792E, M779N, G27D, K955R, 5867R, R693I, F189Y, V635M, F399L, E498K, E3865, V254G, P7935, K188E, QT945KI, T620P, T946P, TT949PP, N952T, K682E, K975R, L212P, E292R, 1303K, C349E, E385P, E386N, D387K, L404K, E466H, C477Q, C477H, C479A, D659H, T806V, K8085, AA5797, V959M, K975Q, W974G, A708Q, V711K, D733T, L742W, V747K, F755M, M771A, M771Q, W782Q, G791F, L792D, L792K, P793Q, P793G, Q804A, Y966N, Y723N, Y857R, 5890R, 5932M, L897M, R624G, 5603G, N7375, L307K, I658V APT688, A5A794, 5877R, N580T, V335G, T6205, W345G, T2805, L406P, A612D, A7515, E386R, V351M, K210N, D40A, E773G, H207L, T62A, T287P, T832A, A8935, AV14, AAG13, R11V, R12N, R13H, AY13, R12L, AQ13,V155,1317. A
indicate insertions, H indicate deletions.

[0028] FIG. 11 is a plot showing individual beneficial mutations can be combined (sometimes referred to as "stacked") for even greater improvements in gene editing activity. CasX proteins were tested for their ability to cleave at 2 different target sites in human HEK293 cells using the E6 and E7 spacers targeting an EGFP reporter, as described in Example 5. The variants, from left to right, are: 5794R + Y797L, K416E+A708K, A708K1P793], [P793]+P793A5, Q367K+14255, A708K1P793]+A793V, Q338R+A339E, Q338R+A339K, 5507G+G508R, L379R+A708K1P793], C477K+A708K1P793], L379R+C477K+A708K1P793], L379R+A708K1P793]+A739V, C477K+A708K1P793]+A739V, L379R+C477K+A708K1P793]+A739V, L379R+A708K1P793]+M779N, L379R+A708K1P793]+M771N, L379R+A708K1P793]+D4895, L379R+A708K1P793]+A739T, L379R+A708K1P793]+D732N, L379R+A708K1P793]+G791M, L379R+A708K1P793]+Y797L, L379R+C477K+A708K1P793]+M779N, L379R+C477K+A708K1P793]+M771N, L379R+C477K+A708K1P793]+D489S, L379R+C477K+A708K1P793]+A739T, L379R+C477K+A708K1P793]+D732N, L379R+C477K+A708K1P793]+G791M, L379R+C477K+A708K1P793]+Y797L, L379R+C477K+A708K1P793]+T620P, A708K1P793]+E386S, E386R+F399L+[P793] and R4581I+A739V of the reference CasX
protein of SEQ ID NO: 2. [] refer to deleted amino acid residues at the specified position of SEQ
ID NO: 2.

[0029] FIG. 12A and FIG. 12B are a pair of plots showing that CasX protein and sgNA
variants when combined, can improve activity more than 6-fold relative to a reference sgRNA
and reference CasX protein pair. sgNA:protein pairs were assayed for their ability to cleave a GFP reporter in HEK293 cells, as described in Example 5. On the Y-axis, the fraction of cells in which expression of the GFP reporter was disrupted by CasX mediated gene editing are shown.
FIG. 12A shows CasX protein and sgNAs that were assayed with the E6 spacer targeting GFP.
FIG. 12B shows CasX protein and sgNAs that were assayed with the E7 spacer targeting GFP.
iGFP stands for "inducible GFP."

[0030] FIG. 13A, FIG. 13B and FIG. 13C show that making and screening DME
libraries has allowed for generation and identification of variants that exhibit a 1 to 81-fold improvement in editing efficiency, as described in Examples 1 and 3. FIG. 13A shows an RFP+
and GFP+
reporter in E. coil cells assayed for CRISPR interference repression of GFP
with a reference nuclease dead CasX protein and sgNA. FIG. 13B shows the same reporter cells assayed for GFP
repression with nuclease dead CasX variants screened from a DME library. FIG.
13C shows improved editing efficiency of a selected CasX protein and sgNA variant compared to the reference with 5 spacers targeting the endogenous B2M locus in HEK 293 human cells. The Y
axis shows disruption in B2M staining by HLA1 antibody indicating gene disruption via CasX
editing and indel formation. The improved CasX variants improved editing of this locus up to 81-fold over the reference in the case of guide spacer # 43. CasX pairs with the reference sgRNA: protein pair of SEQ ID NO: 5 and SEQ ID NO: 2, and CasX variant protein of L379R+A708K1P793] of SEQ ID NO: 2, assayed with the sgNA variant with a truncated stem loop and a T10C substitution, which is encoded by a sequence of TACTGGCGCCTTTATCTCATTACTTTGAGAGCCATCACCAGCGACTATGTCGTATGG

GTAAAGCGCTTACGGACTTCGGTCCGTAAGAAGCATCAAAG (SEQ ID NO: 23), are indicated. The following spacer sequences were used: #9: GTGTAGTACAAGAGATAGAA
(SEQ ID NO: 24); #14: TGAAGCTGACAGCATTCGGG (SEQ ID NO: 25), #20:
tagATCGAGACATGTAAGCA (SEQ ID NO: 26); #37: GGCCGAGATGTCTCGCTCCG
(SEQ ID NO: 27) and #43: AGGCCAGAAAGAGAGAGTAG (SEQ ID NO: 28).

[0031] FIGS. 14A-14F are a series of structural models of a prototypic CasX
protein showing the location of mutations in CasX variant proteins of the disclosure which exhibit improved activity. FIG. 14A shows a deletion of P at 793 of SEQ ID NO: 2, with a deletion in a loop that may affect folding. FIG. 14B shows a replacement of Alanine (A) by Lysine (K) at position 708 of SEQ ID NO: 2. This mutation is facing the gNA 5' end plus a salt bridge to the gNA. FIG.
14C shows a replacement of Cysteine (C) by Lysine (K) at position 477 of SEQ
ID NO: 2. This mutation is facing the gNA. There is salt bridge to the gNAbb (gNA phosphase backbone) at approximately base 14 that may be affected. This mutation removes a surface exposed cysteine.
FIG. 14D shows a replacement of Leucine (L) with Arginine (R) at position 379 of SEQ ID NO:
2. There is a salt bridge to the target DNAbb (DNA phosphate backbone) towards base pairs 22-23 that may be affected. FIG. 14E shows one view of a combination of the deletion of P at 793 and the A708K substitution. FIG. 14F shows an alternate view, that shows that the effects of individual mutants are additive and single mutants can be combined (stacked) for even greater improvements. Arrows indicate the locations of mutations throughout FIG. 14A-14F.

[0032] FIG. 15 is a plot showing the identification of optimal Planctomycetes CasX PAM and spacers for genes of interest, as described in Example 6. On the Y-axis, percent GFP negative cells, indicating cleavage of a GFP reporter, is shown. On the X-axis, different PAM sequences and spacers: ATC PAM, CTC PAM and TTC PAM. GTC, TTT and CTT PAMs were also tested and showed no activity.

[0033] FIG. 16 is a plot showing that improved CasX variants generated by DME
can edit both canonical and non-canonical PAMs more efficiently than reference CasX
proteins, as described in Example 6. The Y-axis shows the average fold improvement in editing relative to a reference sgRNA: protein pair (SEQ ID NO:2, SEQ ID NO: 5) with 2 targets, N=
6. Protein variants, from left to right for each set of bars were: A708K+[P793]+ A739V;
L379R+A708K1P793]; C477K+A708K1P793]; L379R+C477K+A708K1P793];
L379R+A708K1P793]+A739V; C477K+A708K1P793]+A739V; and L379R+C477K+A708K1P793]+A739V. Reference CasX and protein variants were assayed with a reference sgRNA scaffold of SEQ ID NO: 5 with DNA encoding spacer sequences of, from left to right, E6 (SEQ ID NO: 29) with a TTC PAM; E7 (SEQ ID NO: 30) with a TTC
PAM; GFP8 (SEQ ID NO: 31) with a TTC PAM; B1 (SEQ ID NO: 32) with a CTC PAM
and A7 (SEQ ID NO: 33) with an ATC PAM.

[0034] FIGS. 17A-17F are a series of plots showing that a reference CasX
protein and a reference sgRNA scaffold pair is highly specific for the target sequence, as described in Example 7. FIG. 17A and FIG. 17D, Streptococcus pyogenes Cas9 (SpyCas9) was assayed with two different gNA spacers and a 5' PAM site (SEQ ID NOs: 34-65) and (SEQ ID
NOs: 136-166) for its ability to edit templates with a target sequence complementary to the spacer sequence (arrow), or with 1, 2, 3 or 4 mutations in the target sequence relative to the spacer sequence. FIG. 17B and FIG. 17E, Staphylococcus aureus Cas9 (SauCas9) was assayed with two different gNA spacers and a 5' PAM site (SEQ ID NOs: 66-103) and (SEQ ID NOs:
167-204) for its ability to edit templates with a target sequence complementary to the spacer sequence (arrow), or with 1, 2, 3 or 4 mutations in the target sequence relative to the spacer sequence.
FIG. 17C and FIG. 17F, the reference Plm CasX protein and sgNA scaffold pair was assayed with two different gNA spacers and a 3' PAM site (SEQ ID NOs: 104-135) and (SEQ ID NOs:
205-236) for its ability to edit templates with a target sequence complementary to the spacer sequence (arrow), or with 1, 2, 3 or 4 mutations in the target sequence relative to the spacer sequence. In all of FIG. 17A-17F, the X-axis shows the fraction of cells where gene editing at the target sequence occurred.

[0035] FIG. 18 illustrates a scaffold stem loop of an exemplary reference sgRNA of the disclosure (SEQ ID NO: 237).

[0036] FIG. 19 illustrates an extended stem loop sequence of an exemplary reference sgRNA
of the disclosure (SEQ ID NO: 238).

[0037] FIGS. 20A-20B are a pair of plots that demonstrate that specific subsets of changes discovered by DME of the CasX are more likely to predict improvements of activity, as described in Example 4. The plots represent data from the experiments described in FIG.7 and FIG. 8. FIG 20A shows that changing amino acids within a distance of 10 Angstroms (A) of the guide RNA to hydrophobic residues (A, V, I, L, M, F, Y, W) results in a significantly less active protein. FIG. 20B demonstrates that, in contrast, changing a residue within 10 A of the RNA to a positively charged amino acid (R, H, K) is likely to improve activity.

[0038] FIG. 21 illustrates an alignment of two reference CasX protein sequences (SEQ ID
NO: 1, top; SEQ ID NO: 2, bottom), with domains annotated.

[0039] FIG. 22 illustrates the domain organization of a reference CasX protein of SEQ ID NO:
1. The domains have the following coordinates: non-target strand binding (NTSB) domain:
amino acids 101-191; Helical I domain: amino acids 57-100 and 192-332; Helical II domain:
333-509; oligonucleotide binding domain (OBD): amino acids 1-56 and 510-660;
RuvC DNA
cleavage domain (RuvC): amino acids 551-824 and 935-986; target strand loading (TSL) domain: amino acids 825-934. Note that the Helical I, OBD and RuvC domains are non-contiguous.

[0040] FIG. 23 illustrates an alignment of two CasX reference sgRNA scaffolds SEQ ID NO:
(top) and SEQ ID NO: 4 (bottom).

[0041] FIG. 24 shows an SDS-PAGE gel of StX2 (CasX reference of SEQ ID NO: 2) purification fractions visualized by colloidal Coomassie staining, as described in Example 8. The lanes, from left to right, are: Pellet: insoluble portion following cell lysis, Lysate: soluble portion following cell lysis, Flow Thru: protein that did not bind the heparin column, Wash: protein that eluted from the column in wash buffer, Elution: protein eluted from the heparin column with elution buffer, Flow Thru: Protein that did not bind the StrepTactin column, Elution: protein eluted from the StrepTactin column with elution buffer, Injection:
concentrated protein injected onto the s200 gel filtration column, Frozen: pooled fractions from the s200 elution that have been concentrated and frozen.

[0042] FIG. 25 shows the chromatogram from a size exclusion chromatography assay of the StX2, as described in Example 8.

[0043] FIG. 26 shows an SDS-PAGE gel of StX2 purification fractions visualized by colloidal Coomassie staining, as described in Example 8. From right to left: Injection sample, molecular weight markers, lanes 3 -9: samples from the indicated elution volumes.

[0044] FIG. 27 shows the chromatogram from a size exclusion chromatography assay of the CasX 119, using of Superdex 200 16/600 pg gel filtration, as described in Example 8. The 67.47 mL peak corresponds to the apparent molecular weight of CasX variant 119 and contained the majority of CasX variant 119 protein.

[0045] FIG. 28 shows an SDS-PAGE gel of CasX 119 purification fractions visualized by colloidal Coomassie staining, as described in Example 8. Samples from the indicated fractions were resolved by SDS-PAGE and stained with colloidal Coomassie. From right to left, Injection:

sample of protein injected onto the gel filtration column, molecular weight markers, lanes 3 -10:
samples from the indicated elution volumes.

[0046] FIG. 29 shows an SDS-PAGE gel of purification samples of CasX 438, visualized on a Bio-Rad StainFreeTM gel. The lanes, from left to right, are: Pellet: insoluble portion following cell lysis, Lysate: soluble portion following cell lysis, Flow Thru: protein that did not bind the heparin column, Elution: protein eluted from the heparin column with elution buffer, Flow Thru:
Protein that did not bind the StrepTactin column, Elution: protein eluted from the StrepTactin column with elution buffer, Injection: concentrated protein injected onto the s200 gel filtration column, Pool: pooled CasX-containing fractions, Final: pooled fractions from the s200 elution that have been concentrated and frozen.

[0047] FIG. 30 shows the chromatogram from a size exclusion chromatography assay of the CasX 438, using of Superdex 200 16/600 pg gel filtration, as described in Example 8. The 69.13 mL peak corresponds to the apparent molecular weight of CasX variant 438 and contained the majority of CasX variant 438 protein.

[0048] FIG. 31 shows an SDS-PAGE gel of CasX 438 purification fractions visualized by colloidal Coomassie staining, as described in Example 8. Samples from the indicated fractions were resolved by SDS-PAGE and stained with colloidal Coomassie. From right to left, Injection:
sample of protein injected onto the gel filtration column, molecular weight markers, lanes 3 -10:
samples from the indicated elution volumes.

[0049] FIG. 32 shows an SDS-PAGE gel of purification samples of CasX 457, visualized on a Bio-Rad StainFreeTM gel. The lanes, from left to right, are: Pellet: insoluble portion following cell lysis, Lysate: soluble portion following cell lysis, Flow Thru: protein that did not bind the heparin column, Wash, Elution: protein eluted from the heparin column with elution buffer, Flow Thru: Protein that did not bind the StrepTactin column, Elution: protein eluted from the StrepTactin column with elution buffer, Injection: concentrated protein injected onto the s200 gel filtration column, Final: pooled fractions from the s200 elution that have been concentrated and frozen.

[0050] FIG. 33 shows the chromatogram from a size exclusion chromatography assay of the CasX 457, using of Superdex 200 16/600 pg gel filtration, as described in Example 8. The 67.52 mL peak corresponds to the apparent molecular weight of CasX variant 457 and contained the majority of CasX variant 457 protein.

[0051] FIG. 34 shows an SDS-PAGE gel of CasX 457 purification fractions visualized by colloidal Coomassie staining, as described in Example 8. Samples from the indicated fractions were resolved by SDS-PAGE and stained with colloidal Coomassie. From right to left, Injection:
sample of protein injected onto the gel filtration column, molecular weight markers, lanes 3 -10:
samples from the indicated elution volumes.

[0052] FIG. 35 is a schematic showing the organization of the components in the pSTX34 plasmid used to assemble the CasX constructs, as described in Example 9.

[0053] FIG. 36 is a schematic showing the steps of generating the CasX 119 variant, as described in Example 9.

[0054] FIG. 37 is a graph of the results of an assay for the quantification of active fractions of RNP formed by sgRNA174 and the CasX variants 119 and 457, as described in Example 19.
Equimolar amounts of RNP and target were co-incubated and the amount of cleaved target was determined at the indicated timepoints. Mean and standard deviation of three independent replicates are shown for each timepoint. The biphasic fit of the combined replicates is shown.
"2" refers to the reference CasX protein of SEQ ID NO: 2.

[0055] FIG. 38 is a graph of the results of an assay for quantification of active fractions of RNP formed by CasX2 and reference guide 2 the modified sgRNA guides 32, 64, and 174, as described in Example 19. Equimolar amounts of RNP and target were co-incubated and the amount of cleaved target was determined at the indicated timepoints. Mean and standard deviation of three independent replicates are shown for each timepoint. The biphasic fit of the combined replicates is shown. "2" refers to reference gRNAs SEQ ID NO: 5, respectively, and the identifying number of modified sgRNAs are indicated in Table 2.

[0056] FIG. 39 is a graph of the results of an assay for quantification of cleavage rates of RNP
formed by sgRNA174 and the CasX variants 119 and 457, as described in Example 19. Target DNA was incubated with a 20-fold excess of the indicated RNP and the amount of cleaved target was determined at the indicated time points. Mean and standard deviation of three independent replicates are shown for each timepoint. The monophasic fit of the combined replicates is shown.

[0057] FIG. 40 is a graph of the results of an assay for quantification of cleavage rates of RNP
formed by CasX2 and the sgRNA guide variants 2, 32, 64 and 174, as described in Example 19.
Target DNA was incubated with a 20-fold excess of the indicated RNP and the amount of cleaved target was determined at the indicated time points. Mean and standard deviation of three independent replicates are shown for each timepoint. The monophasic fit of the combined replicates is shown.

[0058] FIG. 41 is a graph of the results of an assay for quantification of initial velocities of RNP formed by CasX2 and the sgRNA guide variants 2, 32, 64 and 174, as described in Example 19. The first two time-points of the previous cleavage experiment were fit with a linear model to determine the initial cleavage velocity.

[0059] FIG. 42 is a schematic showing an example of CasX protein and scaffold DNA
sequence for packaging in adeno-associated virus (AAV), as described in Example 20. The DNA
segment between the AAV inverted terminal repeats (ITRs), comprised of a CasX-encoding DNA and its promoter, and scaffold-encoding DNA and its promoter gets packaged within an AAV capsid during AAV production.

[0060] FIG. 43 is a graph showing representative results of AAV titering by qPCR, as described in Example 20. During AAV purification, flow through (FT) and consecutive eluent fractions (1-6) are collected and titered by qPCR. Most virus, ¨1e14 viral genomes in this example, is found in the second elution fraction.

[0061] FIG. 44 shows the results of an AAV-mediated gene editing experiment in the SOD1-GFP reporter cell line, as described in Example 21. CasX constructs (CasX 119 and guide 64 with SOD1 targeting spacer 2, ATGTTCATGAGTTTGGAGAT; SEQ ID NO: 239) and SauCas9 with SOD1 targeting spacer were packaged in AAV vectors and used to transduce SOD1-GFP reporter cells at a range of different multiplicity of infection (MOTs, no. of viral genomes/cell). Twelve days later, cells were assayed for GFP disruption via FACS. In this example, CasX and SauCas9 shows equivalent levels of editing, where 1-2% of the cells show GFP disruption at the highest MOIs, 1e7 or 1e6.

[0062] FIG. 45 shows the results of a second AAV-mediated gene editing experiment in the SOD1-GFP reporter cell line, as described in Example 21. CasX constructs 119.64 with SOD1 targeting spacer (2, ATGTTCATGAGTTTGGAGAT; SEQ ID NO: 239) and SauCas9 with SOD1 targeting spacer were packaged in AAV vectors and used to transduce SOD1-GFP
reporter cells at a range of different multiplicity of infection (MOTs, no. of viral genomes/cell).
Twelve days later, cells were assayed for GFP disruption via FACS. In this example, CasX and SauCas9 shows equivalent levels of editing at the highest MOI, where ¨2-4% of the cells show GFP disruption.

[0063] FIG. 46 shows the results of an AAV-mediated gene editing experiment in neural progenitor cells (NPCs) from the G93A mouse model of ALS, as described in Example 21.
CasX constructs (CasX 119 and guide 64 with SOD1 targeting spacer 2, ATGTTCATGAGTTTGGAGAT; SEQ ID NO: 239) was packaged in an AAV vector and used to transduce G93A NPCs at a range of different multiplicity of infection (MOIs, no. of viral genomes/cell). Twelve days later, cells were assayed for gene editing via T7E1 assay. Agarose gel image from the T7E1 assay shown here demonstrates successful editing of the SOD1 locus.
Double arrows show the two DNA bands as a result of successful editing in cells.

[0064] FIG. 47 shows the results of an editing assay of 6 target genes in HEK293T cells, as described in Example 23. Each dot represents results using an individual spacer.

[0065] FIG. 48 shows the results of an editing assay of 6 target genes in HEK293T cells, with individual bars representing the results obtained with individual spacers, as described in Example 23.

[0066] FIG. 49 shows the results of an editing assay of 4 target genes in HEK293T cells, as described in Example 23. Each dot represents results using an individual spacer utilizing a CTC
(CTCN) PAM.

[0067] FIG. 50 is a schematic showing the steps of Deep Mutational Evolution used to create libraries of genes encoding CasX variants, as described in Example 24. The pSTX1 backbone is minimal, composed of only a high-copy number origin and KanR resistance gene, making it compatible with the recombineering E. coli strain EcNR2. pSTX2 is a BsmbI
destination plasmid for aTc-inducible expression in E. coli.

[0068] FIG. 51 are dot plot graphs showing the results of CRISPRi screens for mutations in libraries D1, D2, and D3, as described in Example 24. In the absence of CRISPRi, E. coli constitutively express both GFP and RFP, resulting in intense fluorescence in both wavelengths, represented by dots in the upper-right region of the plot. CasX proteins resulting in CRISPRi of GFP can reduce green fluorescence by >10-fold, while leaving red fluorescence unaltered, and these cells fall within the indicated Sort Gate 1. The total fraction of cells exhibiting CRISPRi is indicated.

[0069] FIG. 52 are photographs of colonies grown in the ccdB assay, as described in Example 24. 10-fold dilutions were assayed in the presence of glucose or arabinose to induce expression of the ccdB toxin, resulting in approximately a 1000-fold difference between functional and nonfunctional proteins. When grown in liquid culture, the resolving power was approximately 10,000-fold, as seen on the right-hand side.

[0070] FIG. 53 is a graph of HEK iGFP genome editing efficiency testing CasX
variants with sgRNA 2 (SEQ ID NO:5), with appropriate spacers, with data expressed as fold-improvement over the wild-type CasX protein (SEQ ID NO: 2) in the HEK iGFP editing assay, as described in Example 24. Single mutations are shown at the top, with groups of mutations shown at the bottom of the graph). Error bars combine internal measurement error (SD) and inter-experimental measurement error (SD across replicate experiments for those variants tested more than once), in at least triplicate assays.

[0071] FIG. 54 is a scatterplot showing results of the SOD1-GFP reporter assay for CasX
variants with sgRNA scaffold 2 utilizing two different spacers for GFP, as described in Example 24.

[0072] FIG. 55 is a graph showing the results of the HEK293 iGFP genome editing assay assessing editing across four different PAM sequences comparing wild-type CasX
(SEQ ID NO:
2) and CasX variant 119; both utilizing sgRNA scaffold 1 (SEQ ID NO: 4), with spacers utilizing four different PAM sequences, as described in Example 24.

[0073] FIG. 56 is a graph showing the results of genome editing activity of CasX variant 119 and sgRNA 174 compared to wild-type CasX 2 and guide scaffold 1 in the iGFP
lipofection assay utilizing two different spacers, as described in Example 24.

[0074] FIG. 57 is a graph showing the results of genome editing activity of CasX variant 119 and sgRNA 174 compared to wild-type CasX and guide in the iGFP lentiviral transduction assay, using two different spacers, as described in Example 24.

[0075] FIG. 58 is a graph showing the results of genome editing in the more stringent lentiviral assay to compare the editing activity of four CasX variants (119, 438, 488 and 491) and the optimized sgNA 174 and two different spacers, as described in Example 24. The results show the step-wise improvement in editing efficiency achieved by the additional modifications and domain swaps introduced to the starting-point 119 variant.

[0076] FIGS. 59A- 59B shows the results of NGS analyses of the libraries of sgRNA, as described in Example 25. FIG. 59A shows the distribution of substitutions, deletions and insertions. FIG. 59B is a scatterplot showing the high reproducibility of variant representation in two separate library pools after the CRISPRi assay in the unsorted, naive population of cells.

(Library pool D3 vs D2 are two different versions of the dCasX protein, and represent replicates of the CRISPRi assay.)

[0077] FIGS. 60A-60B shows the structure of wild-type CasX and RNA guide (SEQ
ID
NO:4). FIG. 60A depicts the CryoEM structure of Deltaproteobacteria CasX
protein:sgRNA
RNP complex (PDB id: 6YN2), including two stem loops, a pseudoknot, and a triplex. FIG. 60B
depicts the secondary structure of the sgRNA was identified from the structure shown in (A) using the tool RNAPDBee 2.0 (rnapdbee.cs.put.poznan.p1/, using the tools 3DNA/DSSR, and using the VARNA visualization tool). RNA regions are indicated. Residues that were not evident in the PDB crystal structure file are indicated by plain-text letters (i.e., not encircled), and are not included in residue numbering.

[0078] FIGS. 61A-61C depicts comparisons between two guide RNA scaffolds. FIG.

provides the sequence alignment between the single guide scaffold 1 (SEQ ID
NO: 4) and scaffold 2 (SEQ ID NO: 5). FIG. 61B shows the predicted secondary structure of scaffold 1 (without the 5' ACAUCU bases which were not in the cryoEM structure).
Prediction was done using RNAfold (v 2.1.7), using a constraint that was derived from the base-pairing observed in the cryoEM structure (see FIGS. 60A-60B). This constraint required the base pairs observed in the cryoEM structure to be formed, and required the bases involved in triplex formation to be unpaired. This structure has distinct base pairing from the lowest-energy predicted structure at the 5' end (i.e., the pseudoknot and triplex loop). FIG. 61C shows the predicted secondary structure of scaffold 2. Prediction was done for scaffold 1, using a similar constraint based on the sequence alignment.

[0079] FIG. 62 shows a graph comparing GFP-knockdown capability of scaffold 1 versus scaffold 2 in GFP-lipofection assay, using four different spacers utilizing different PAM
sequences, as described in Example 25. The results demonstrate the greater editing imparted by use of the modified scaffold 2 compared to the wild-type scaffold 1; the latter showing no editing with spacers utilizing GTC and CTC PAM sequences.

[0080] FIGS. 63A-63C shows graphs depicting the enrichment of single variants across the scaffold, revealing mutable regions, as described in Example 25. FIG. 63A
depicts substituted bases (A, T, G, or C; top to bottom), FIG. 63B depicts inserted bases (A, T, G, or C; top to bottom), and FIG. 63C depicts deletions at the individual nucleotide position (X-axis) across scaffold 2. Enrichment values were averaged across the three dead CasX
versions, relative to the average WT value. Scaffolds with relative 1og2 enrichment > 0 are considered 'enriched', as they were more represented in the sorted population relative to the naive population than the wildtype scaffold was represented. Error bars represent the confidence interval across the three catalytically dead CasX experiments.

[0081] FIG. 64 are scatterplots showing that the enrichment values obtained across different dCasX variants are largely consistent, as described in Example 25. Libraries D2 and DDD have highly correlated enrichment scores, while D3 is more distinct.

[0082] FIG. 65 shows a bar graph of cleavage activity of several scaffold variants in a more stringent lipofection assay at the SOD1-GFP locus, as described in Example 25.

[0083] FIG. 66 shows a bar graph of cleavage activity for several scaffold variants using two different spacers; 8.2 and 8.4 that target SOD1-GFP locus (and a non-targeting spacer NT), with low-MOI lentiviral transduction using a p34 plasmid backbone, as described in Example 25.

[0084] FIG. 67 is a schematic showing the secondary structure of single guide 174 on top and the linear structure on the bottom, with lines joining those segments associating by base-pairing or other non-covalent interactions. The scaffold stem (white, no fill) (and loop) and the extended stem (grey, no fill) (and loop) are adjacent from 5' to 3' in the sequence. However, the pseudoknot and extended stems are formed from strands that have intervening regions in the sequence. The triplex is formed, in the case of single guide 174, comprising nucleotides 5'-CUUUG'-3' AND 5' -CAAAG-3' that form a base-paired duplex and nucleotides 5'-UUU-3' that associates with the 5'-AAA-3' to form the triplex region.

[0085] FIG. 68 shows comparisons between the highly-evolved single guide 174 and the scaffolds 1 and 2 that served as the starting points for the DME procedures described in Example 25. FIG. 68A shows a bar graph of cleavage activity of head-to-head comparisons of cleavage activity of the guide scaffolds with five different spacers in a plasmid lipofection assay at the GFP locus in HEK-GFP cells. FIG. 68B shows the sequence alignment between scaffold 2 and guide 174 (SEQ ID NO: 2238). Asterisks indicate point mutations, and the dotted box shows the entire extended stem swap.

[0086] FIGS. 69A-69B shows scatterplots of HEK-iGFP cleavage assay for scaffolds sequences relative to WT scaffold with 2 spacers; 4.76 (FIG. 69A) and 4.77 (FIG. 69B), as described in Example 25.

[0087] FIG. 70 shows a scatterplot comparing the normalized cleavage activity of several scaffolds relative to WT with 2 spacers (4.76 and 4.77), as described in Example 25. Error bars combine internal measurement error (SD) and inter-experimental measurement error (SD across replicate experiments for those variants tested more than once), in quadrature.

[0088] FIG. 71 shows a scatterplot comparing the normalized cleavage activity of multiple scaffolds relative to WT in the HEK-iGFP cleavage assay to the enrichments obtained from the CRISPRi comprehensive screen, as described in Example 25. Generally, scaffold mutations with high enrichment (>1.5) have cleavage activity comparable to or greater than WT. Two variants have high cleavage activity with low enrichment scores (C18G and T17G);
interestingly, these substitutions are at the same position as several highly enriched insertions (FIGS. 63A-63C).
Labels indicate the mutations for a subset of the comparisons.

[0089] FIG. 72 shows the results of flow cytometry analysis of Cas-mediated editing at the RHO locus in APRE19 RHO-GFP cells 14 days post-transfection for the CasX
variant constructs 438, 499 and 491, as described in Example 26. The points are the results of individual samples and the light dashed lines are upper and lower quartiles.

[0090] FIG. 73 shows the quantification of cleavage rates of RNP formed by sgRNA174 and the CasX variants on targets with different PAMs. Target DNA was incubated with a 20-fold excess of the indicated RNP and the amount of cleaved target was determined at the indicated time points. The monophasic fit of the combined replicates is shown.
DETAILED DESCRIPTION

[0091] While exemplary embodiments have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only.
Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the inventions claimed herein. It should be understood that various alternatives to the embodiments described herein may be employed in practicing the embodiments of the disclosure. It is intended that the claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
Defintions

[0092] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present embodiments, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control.

In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention.

[0093] The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides.
Thus, terms "polynucleotide" and "nucleic acid" encompass single-stranded DNA;
double-stranded DNA; multi-stranded DNA; single-stranded RNA; double-stranded RNA;
multi-stranded RNA; genomic DNA; cDNA; DNA-RNA hybrids; and a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0094] "Hybridizable" or "complementary" are used interchangeably to mean that a nucleic acid (e.g., RNA, DNA) comprises a sequence of nucleotides that enables it to non-covalently bind, i.e., form Watson-Crick base pairs and/or G/U base pairs, "anneal", or "hybridize," to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and/or in vivo conditions of temperature and solution ionic strength. It is understood that the sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable; it can have at least about 70%, at least about 80%, or at least about 90%, or at least about 95% sequence identity and still hybridize to the target nucleic acid.
Moreover, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure, a 'bulge', 'bubble' and the like).

[0095] A "gene," for the purposes of the present disclosure, includes a DNA
region encoding a gene product (e.g., a protein, RNA), as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. Accordingly, a gene may include regulatory sequences including, but not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions.
Coding sequences encode a gene product upon transcription or transcription and translation; the coding sequences of the disclosure may comprise fragments and need not contain a full-length open reading frame. A gene can include both the strand that is transcribed, e.g. the strand containing the coding sequence, as well as the complementary strand.

[0096] The term "downstream" refers to a nucleotide sequence that is located 3' to a reference nucleotide sequence. In certain embodiments, downstream nucleotide sequences relate to sequences that follow the starting point of transcription. For example, the translation initiation codon of a gene is located downstream of the start site of transcription.

[0097] The term "upstream" refers to a nucleotide sequence that is located 5' to a reference nucleotide sequence. In certain embodiments, upstream nucleotide sequences relate to sequences that are located on the 5' side of a coding region or starting point of transcription. For example, most promoters are located upstream of the start site of transcription.

[0098] The term "regulatory element" is used interchangeably herein with the term "regulatory sequence," and is intended to include promoters, enhancers, and other expression regulatory elements (e.g. transcription termination signals, such as polyadenylation signals and poly-U sequences). Exemplary regulatory elements include a transcription promoter such as, but not limited to, CMV, CMV+intron A, SV40, RSV, HIV-Ltr, elongation factor 1 alpha (EF1a), MMLV-ltr, internal ribosome entry site (IRES) or P2A peptide to permit translation of multiple genes from a single transcript, metallothionein, a transcription enhancer element, a transcription termination signal, polyadenylation sequences, sequences for optimization of initiation of translation, and translation termination sequences. It will be understood that the choice of the appropriate regulatory element will depend on the encoded component to be expressed (e.g., protein or RNA) or whether the nucleic acid comprises multiple components that require different polymerases or are not intended to be expressed as a fusion protein.

[0099] The term "promoter" refers to a DNA sequence that contains an RNA
polymerase binding site, transcription start site, TATA box, and/or B recognition element and assists or promotes the transcription and expression of an associated transcribable polynucleotide sequence and/or gene (or transgene). A promoter can be synthetically produced or can be derived from a known or naturally occurring promoter sequence or another promoter sequence. A
promoter can be proximal or distal to the gene to be transcribed. A promoter can also include a chimeric promoter comprising a combination of two or more heterologous sequences to confer certain properties. A promoter of the present disclosure can include variants of promoter sequences that are similar in composition, but not identical to, other promoter sequence(s) known or provided herein. A promoter can be classified according to criteria relating to the pattern of expression of an associated coding or transcribable sequence or gene operably linked to the promoter, such as constitutive, developmental, tissue-specific, inducible, etc.

[00100] The term "enhancer" refers to regulatory element DNA sequences that, when bound by specific proteins called transcription factors, regulate the expression of an associated gene.
Enhancers may be located in the intron of the gene, or 5' or 3' of the coding sequence of the gene. Enhancers may be proximal to the gene (i.e., within a few tens or hundreds of base pairs (bp) of the promoter), or may be located distal to the gene (i.e., thousands of bp, hundreds of thousands of bp, or even millions of bp away from the promoter). A single gene may be regulated by more than one enhancer, all of which are envisaged as within the scope of the instant disclosure.

[00101] "Recombinant," as used herein, means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, and/or ligation steps resulting in a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems. Generally, DNA sequences encoding the structural coding sequence can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of synthetic oligonucleotides, to provide a synthetic nucleic acid which is capable of being expressed from a recombinant transcriptional unit contained in a cell or in a cell-free transcription and translation system. Such sequences can be provided in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, which are typically present in eukaryotic genes. Genomic DNA comprising the relevant sequences can also be used in the formation of a recombinant gene or transcriptional unit. Sequences of non-translated DNA
may be present 5' or 3' from the open reading frame, where such sequences do not interfere with manipulation or expression of the coding regions, and may indeed act to modulate production of a desired product by various mechanisms (see "enhancers" and "promoters", above).

[00102] The term "recombinant polynucleotide" or "recombinant nucleic acid"
refers to one which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of sequence through human intervention. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Such can be done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a desired combination of functions. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.

[00103] Similarly, the term "recombinant polypeptide" or "recombinant protein"
refers to a polypeptide or protein which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of amino sequence through human intervention. Thus, e.g., a protein that comprises a heterologous amino acid sequence is recombinant.

[00104] As used herein, the term "contacting" means establishing a physical connection between two or more entities. For example, contacting a target nucleic acid with a guide nucleic acid means that the target nucleic acid and the guide nucleic acid are made to share a physical connection; e.g., can hybridize if the sequences share sequence similarity.

[00105] "Dissociation constant", or "Kd", are used interchangeably and mean the affinity between a ligand "L" and a protein "P"; i.e., how tightly a ligand binds to a particular protein. It can be calculated using the formula Kd=[L] [P]/[LP], where [P], [L] and [LP]
represent molar concentrations of the protein, ligand and complex, respectively.

[00106] The disclosure provides compositions and methods useful for editing a target nucleic acid sequence. As used herein "editing" is used interchangeably with "modifying" and includes but is not limited to cleaving, nicking, deleting, knocking in, knocking out, and the like.

[00107] As used herein, "homology-directed repair" (HDR) refers to the form of DNA repair that takes place during repair of double-strand breaks in cells. This process requires nucleotide sequence homology, and uses a donor template to repair or knock-out a target DNA, and leads to the transfer of genetic information from the donor (e.g., such as the donor template) to the target.
Homology-directed repair can result in an alteration of the sequence of the target nucleic acid sequence by insertion, deletion, or mutation if the donor template differs from the target DNA
sequence and part or all of the sequence of the donor template is incorporated into the target DNA at the correct genomic locus.

[00108] As used herein, "non-homologous end joining" (NHEJ) refers to the repair of double-strand breaks in DNA by direct ligation of the break ends to one another without the need for a homologous template (in contrast to homology-directed repair, which requires a homologous sequence to guide repair). NHEJ often results in indels; the loss (deletion) or insertion of nucleotide sequence near the site of the double- strand break.

[00109] As used herein "micro-homology mediated end joining" (MMEJ) refers to a mutagenic DSB repair mechanism, which always associates with deletions flanking the break sites without the need for a homologous template (in contrast to homology-directed repair, which requires a homologous sequence to guide repair). MMEJ often results in the loss (deletion) of nucleotide sequence near the site of the double- strand break.

[00110] A polynucleotide or polypeptide (or protein) has a certain percent "sequence similarity"
or "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence similarity (sometimes referred to as percent similarity, percent identity, or homology) can be determined in a number of different manners. To determine sequence similarity, sequences can be aligned using the methods and computer programs that are known in the art, including BLAST, available over the world wide web at ncbi.nlm.nih.gov/BLAST. Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined using any convenient method.
Example methods include BLAST programs (basic local alignment search tools) and PowerBLAST
programs (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), e.g., using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl.
Math., 1981, 2, 482-489).

[00111] The terms "polypeptide," and "protein" are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence.

[00112] A "vector" or "expression vector" is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, i.e., an "insert", may be attached so as to bring about the replication or expression of the attached segment in a cell.

[00113] The term "naturally-occurring" or "unmodified" or "wild-type" as used herein as applied to a nucleic acid, a polypeptide, a cell, or an organism, refers to a nucleic acid, polypeptide, cell, or organism that is found in nature.

[00114] As used herein, a "mutation" refers to an insertion, deletion, substitution, duplication, or inversion of one or more amino acids or nucleotides as compared to a wild-type or reference amino acid sequence or to a wild-type or reference nucleotide sequence.

[00115] As used herein the term "isolated" is meant to describe a polynucleotide, a polypeptide, or a cell that is in an environment different from that in which the polynucleotide, the polypeptide, or the cell naturally occurs. An isolated genetically modified host cell may be present in a mixed population of genetically modified host cells.

[00116] A "host cell," as used herein, denotes a eukaryotic cell, a prokaryotic cell, or a cell from a multicellular organism (e.g., a cell line) cultured as a unicellular entity, which cells are used as recipients for a nucleic acid (e.g., an expression vector), and include the progeny of the original cell which has been genetically modified by the nucleic acid. It is understood that the progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation. A "recombinant host cell" (also referred to as a "genetically modified host cell") is a host cell into which has been introduced a heterologous nucleic acid, e.g., an expression vector.

[00117] The term "conservative amino acid substitution" refers to the interchangeability in proteins of amino acid residues having similar side chains. For example, a group of amino acids having aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains consists of serine and threonine; a group of amino acids having amide-containing side chains consists of asparagine and glutamine; a group of amino acids having aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains consists of lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains consists of cysteine and methionine.
Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[00118] As used herein, "treatment" or "treating," are used interchangeably herein and refer to an approach for obtaining beneficial or desired results, including but not limited to a therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder or disease being treated. A therapeutic benefit can also be achieved with the eradication or amelioration of one or more of the symptoms or an improvement in one or more clinical parameters associated with the underlying disease such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder.

[00119] The terms "therapeutically effective amount" and "therapeutically effective dose", as used herein, refer to an amount of a composition, vector, cells, etc., that is capable of having any detectable, beneficial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition when administered in one or repeated doses to a subject. Such effect need not be absolute to be beneficial. Such effect can be transient.

[00120] As used herein, "administering" is meant as a method of giving a dosage of a composition of the disclosure to a subject.

[00121] As used herein, a "subject" is a mammal. Mammals include, but are not limited to, domesticated animals, primates, non-human primates, humans, dogs, porcine (pigs), rabbits, mice, rats and other rodents.

[00122] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
I. General Methods

[00123] The practice of the present invention employs, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift &
Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997);
and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle &
Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference.

[00124] Where a range of values is provided, it is understood that endpoints are included and that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included.

[00125] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[00126] It must be noted that as used herein and in the appended claims, the singular forms "a,"
"an," and "the" include plural referents unless the context clearly dictates otherwise.

[00127] It will be appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. In other cases, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. It is intended that all combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.
CasX:gNA Systems

[00128] In a first aspect, the present disclosure provides CasX:gNA systems comprising a CasX
protein and one or more guide nucleic acids (gNA) for use in modifying or editing a target nucleic acid, inclusive of coding and non-coding regions. The terms CasX
protein and CasX are used interchangeably herein; the terms CasX variant protein and CasX variant are used interchangeably herein. The CasX protein and gNA of the CasX:gNA systems provided herein each independently may be a reference CasX protein, a CasX variant protein, a reference gNA, a gNA variant, or any combination of a reference CasX protein, reference gNA, CasX variant protein, or gNA variant. A gNA and a CasX protein, a gNA variant and CasX
variant, or any combination thereof can form a complex and bind via non-covalent interactions, referred to herein as a ribonucleoprotein (RNP) complex. In some embodiments, the use of a pre-complexed CasX:gNA confers advantages in the delivery of the system components to a cell or target nucleic acid for editing of the target nucleic acid. In the RNP, the gNA can provide target specificity to the RNP complex by including a spacer sequence (targeting sequence) having a nucleotide sequence that is complementary to a sequence of a target nucleic acid. In the RNP, the CasX protein of the pre-complexed CasX:gNA provides the site-specific activity and is guided to a target site (and further stabilized at a target site) within a target nucleic acid sequence to be modified by virtue of its association with the gNA. The CasX protein of the RNP complex provides the site-specific activities of the complex such as binding, cleavage, or nicking of the target sequence by the CasX protein. Provided herein are compositions and cells comprising the reference CasX proteins, CasX variant proteins, reference gNAs, gNA variants, and CasX:gNA
gene editing pairs of any combination of CasX and gNA, as well as delivery modalities comprising the CasX:gNA. In other embodiments, the disclosure provides vectors encoding or comprising the CasX:gNA pair and, optionally, donor templates for the production and/or delivery of the CasX:gNA systems. Also provided herein are methods of making CasX proteins and gNA, as well as methods of using the CasX and gNA, including methods of gene editing and methods of treatment. The CasX proteins and gNA components of the CasX:gNA and their features, as well as the delivery modalities and the methods of using the compositions are described more fully, below.

[00129] The donor templates of the CasX:gNA systems are designed depending on whether they are utilized to correct mutations in a target gene or insert a transgene at a different locus in the genome (a "knock-in"), or are utilized to disrupt the expression of a gene product that is aberrant; e.g., it comprises one or more mutations reducing expression of the gene product or rendering the protein dysfunctional (a "knock-down" or "knock-out"). In some embodiments, the donor template is a single stranded DNA template or a single stranded RNA
template. In other embodiments, the donor template is a double stranded DNA template. In some embodiments, the CasX:gNA systems utilized in the editing of the target nucleic acid comprises a donor template having all or at least a portion of an open reading frame of a gene in the target nucleic acid for insertion of a corrective, wild-type sequence to correct a defective protein. In other cases, the donor template comprises all or a portion of a wild-type gene for insertion at a different locus in the genome for expression of the gene product. In still other cases, a portion of the gene can be inserted upstream (5) of the mutation in the target nucleic acid, wherein the donor template gene portion spans to the C-terminus of the gene, resulting, upon its insertion into the target nucleic acid, in expression of the gene product. In other embodiments, the donor template can comprise one or more mutations in an encoding sequence compared to a normal, wild-type sequence of the target gene utilized for insertion for either knocking out or knocking down (described more fully, below) the defective target nucleic acid sequence.
In other embodiments, the donor template can comprise regulatory elements, an intron, or an intron-exon junction having sequences specifically designed to knock-down or knock-out a defective gene or, in the alternative, to knock-in a corrective sequence to permit the expression of a functional gene product. In some embodiments, the donor polynucleotide comprises at least about 10, at least about 20, at least about 50, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 10,000, at least about 15,000, at least about 25,000, at least about 50,000, at least about 100,000 or at least about 200,000 nucleotides. Provided that there are stretches of DNA sequence with sufficient numbers of nucleotides having sufficient homology flanking the cleavage site(s) of the target nucleic acid sequence targeted by the CasX:gNA (i.e., 5' and 3' to the cleavage site) to support homology-directed repair (the flanking regions being "homologous arms"), use of such donor templates can result in its integration into the target nucleic acid by HDR. In other cases, the donor template can be inserted by non-homologous end joining (NHEJ; which does not require homologous arms) or by microhomology-mediated end joining (MMEJ; which requires short regions of homology on the 5' and 3' ends). In some embodiments, the donor template comprises homologous arms on the 5' and 3' ends, each having at least about 2, at least about 10, at least about 20, at least about 30, at least about 50, at least about 100, at least about 150, at least about 300, at least about 1000, at least about 1500 or more nucleotides having homology with the sequences flanking the intended cleave site(s) of the target nucleic acid. In some embodiments, the CasX:gNA
systems utilize two or more gNA with targeting sequences complementary to overlapping or different regions of the target nucleic acid such that the defective sequence can be excised by multiple double-stranded breaks or by nicking in locations flanking the defective sequence and the donor template inserted by HDR to replace the excised sequence. In the foregoing, the gNA would be designed to contain targeting sequences that are 5' and 3' to the individual site or sequence to be excised. By such appropriate selection of the targeting sequences of the gNA, defined regions of the target nucleic acid can be edited using the CasX:gNA systems described herein.
III. Guide Nucleic Acids of the CasX:gNA Systems

[00130] In other aspects, the disclosure provides guide nucleic acids (gNA) utilized in the CasX:gNA systems, and have utility in editing of a target nucleic acid. The present disclosure provides specifically-designed gNAs with targeting sequences (or "spacers") that are complementary to (and are therefore able to hybridize with) the target nucleic acid as a component of the gene editing CasX:gNA systems. It is envisioned that in some embodiments, multiple gNAs (e.g., multiple gRNAs) are delivered by the CasX:gNA system for the modification of different regions of a gene, including regulatory elements, an exon, an intron, or an intron-exon junction. In some embodiments, the targeting sequence of the gNA is complementary to a sequence comprising one or more single nucleotide polymorphisms (SNPs) of the target nucleic. In other embodiments, the targeting sequence of the gNA
is complementary to a sequence of an intergenic region. For example, when a deletion of a protein-encoding gene is desired, a pair of gNAs with targeting sequences to different or overlapping regions of the target nucleic acid sequence can be used in order to bind and cleave at two different sites within the gene that can then be edited by indel formation or homology-directed repair (HDR), which, in the case of HDR, utilizes a donor template that is inserted to replace the deleted sequence to complete the editing.
a. Reference gNA and gNA variants

[00131] In some embodiments, a gNA of the present disclosure comprises a sequence of a naturally-occurring gNA ("reference gNA"). In other cases, a reference gNA of the disclosure may be subjected to one or more mutagenesis methods, such as the mutagenesis methods described herein, which may include Deep Mutational Evolution (DME), deep mutational scanning (DMS), error prone PCR, cassette mutagenesis, random mutagenesis, staggered extension PCR, gene shuffling, or domain swapping, in order to generate one or more gNA
variants with enhanced or varied properties relative to the reference gNA. gNA
variants also include variants comprising one or more exogenous sequences, for example fused to either the 5' or 3' end, or inserted internally. The activity of reference gNAs may be used as a benchmark against which the activity of gNA variants are compared, thereby measuring improvements in function or other characteristics of the gNA variants. In other embodiments, a reference gNA
may be subjected to one or more deliberate, targeted mutations in order to produce a gNA
variant, for example a rationally-designed variant. As used herein, the terms gNA, gRNA, and gDNA cover naturally-occurring molecules (reference molecules), as well as sequence variants.

[00132] In some embodiments, the gNA is a deoxyribonucleic acid molecule ("gDNA"); in some embodiments, the gNA is a ribonucleic acid molecule ("gRNA"), and in other embodiments, the gNA is a chimera, and comprises both DNA and RNA.

[00133] The gNAs of the disclosure comprise two segments; a targeting sequence and a protein-binding segment (which constitutes the scaffold, discussed herein).
The targeting segment of a gNA includes a nucleotide sequence (referred to interchangeably herein as a guide sequence, a spacer, a targeting sequence, or a targeting region) that is complementary to (and therefore hybridizes with) a specific sequence (a target site) within the target nucleic acid sequence (e.g., a target ssRNA, a target ssDNA, the complementary strand of a double stranded target DNA, etc.), described more fully below.

[00134] The targeting sequence of a gNA is capable of binding to a target nucleic acid sequence, including a coding sequence, a complement of a coding sequence, a non-coding sequence, and to regulatory elements. The protein-binding segment (or "protein-binding sequence") interacts with (e.g., binds to) a CasX protein. The protein-binding segment is alternatively referred to herein as a "scaffold". In some embodiments, the targeting sequence and scaffold each include complementary stretches of nucleotides that hybridize to one another to form a double stranded duplex (e.g. dsRNA duplex for a gRNA). Site-specific binding and/or cleavage of a target nucleic acid sequence (e.g., genomic DNA) by the CasX:gNA
can occur at one or more locations of a target nucleic acid, determined by base-pairing complementarity between the targeting sequence of the gNA and the target nucleic acid sequence.

[00135] The gNA provides target specificity to the complex by having a nucleotide sequence that is complementary to a target sequence of a target nucleic acid. The CasX
of the complex provides the site-specific activities of the complex such as binding, cleavage, or nicking of the target sequence of the target nucleic acid by the CasX nuclease and/or an activity provided by a fusion partner in case of a CasX containing fusion protein, described below.
In some embodiments, the disclosure provides gene editing pairs of a CasX and gNA of any of the embodiments described herein that are capable of being bound together prior to their use for gene editing and, thus, are "pre-complexed" as the RNP. The use of a pre-complexed RNP
confers advantages in the delivery of the system components to a cell or target nucleic acid sequence for editing of the target nucleic acid sequence. The CasX protein of the RNP provides the site-specific activity that is guided to a target site (e.g., stabilized at a target site) within a target nucleic acid sequence by virtue of its association with the guide RNA
comprising a targeting sequence.

[00136] In some embodiments, wherein the gNA is a gRNA, the term "targeter" or "targeter RNA" is used herein to refer to a crRNA-like molecule (crRNA: "CRISPR RNA") of a CasX

dual guide RNA (dgRNA). In a single guide RNA (sgRNA), the "activator" and the "targeter"
are linked together, e.g., by intervening nucleotides). Thus, for example, a guide RNA (dgRNA
or sgRNA) comprises a guide sequence and a duplex-forming segment of a crRNA, which can also be referred to as a crRNA repeat. Because the targeter sequence of a guide sequence hybridizes with a specific target nucleic acid sequence, a targeter can be modified by a user to hybridize with a desired target nucleic acid sequence. In some embodiments, the sequence of a targeter may often be a non-naturally occurring sequence. The targeter and the activator each have a duplex-forming segment, where the duplex forming segment of the targeter and the duplex-forming segment of the activator have complementarity with one another and hybridize to one another to form a double stranded duplex (dsRNA duplex for a gRNA). In some embodiments, a targeter comprises both the guide sequence of the CasX guide RNA and a stretch of nucleotides that forms one half of the dsRNA duplex of the protein-binding segment of the gNA. A corresponding tracrRNA-like molecule (the activator "trans-acting CRISPR RNA") also comprises a duplex-forming stretch of nucleotides that forms the other half of the dsRNA
duplex of the protein-binding segment of the CasX guide RNA. In some cases the activator comprises one or more stem loops that can interact with CasX protein. Thus, a targeter and an activator, as a corresponding pair, hybridize to form a CasX dual guide NA, referred to herein as a "dual guide NA", a "dgNA", a "double-molecule guide NA", or a "two-molecule guide NA".

[00137] In some embodiments, the activator and targeter of the reference gNA
are covalently linked to one another and comprise a single molecule, referred to herein as a "single-molecule guide NA," "one-molecule guide NA," "single guide NA", "single guide RNA", a "single-molecule guide RNA," a "one-molecule guide RNA", a "single guide DNA", a "single-molecule DNA," or a "one-molecule guide DNA", ("sgNA", "sgRNA", or a "sgDNA"). In some embodiments, the sgNA includes an "activator" or a "targeter" and thus can be an "activator-RNA" and a "targeter-RNA," respectively.

[00138] The reference gRNAs of the disclosure comprise four distinct regions, or domains: the RNA triplex, the scaffold stem, the extended stem, and the targeting sequence (specific for a target nucleic acid. The RNA triplex, the scaffold stem, and the extended stem, together, are referred to as the "scaffold" of the reference gNA, based upon which further gNA variants are generated.

b. RNA triplex

[00139] In some embodiments of the guide NAs provided herein, the gNA
comprises an RNA
triplex, and the RNA triplex comprises the sequence of a UUU--Nx(-4-15)--UUU
stem loop (SEQ ID NO: 241) that ends with an AAAG after 2 intervening stem loops (the scaffold stem loop and the extended stem loop), forming a pseudoknot that may also extend past the triplex into a duplex pseudoknot. The UU-UUU-AAA sequence of the triplex forms as a nexus between the targeting sequence, scaffold stem, and extended stem. In exemplary gRNAs, the UUU-loop-UUU region is coded for first, then the scaffold stem loop, and then the extended stem loop, which is linked by the tetraloop, and then an AAAG closes off the triplex before becoming the targeting sequence.
c. Scaffold Stem Loop

[00140] In some embodiments of gNAs of the disclosure, the triplex region is followed by the scaffold stem loop. The scaffold stem loop is a region of the gNA that is bound by CasX protein (such as a reference or CasX variant protein). In some embodiments, the scaffold stem loop is a fairly short and stable stem loop, and increases the overall stability of the gNA. In some cases, the scaffold stem loop does not tolerate many changes, and requires some form of an RNA
bubble. In some embodiments, the scaffold stem is necessary for gNA function.
While it is perhaps analogous to the nexus stem of Cas9 as being a critical stem loop, the scaffold stem of a gNA, in some embodiments, has a necessary bulge (RNA bubble) that is different from many other stem loops found in CRISPR/Cas systems. In some embodiments, the presence of this bulge is conserved across gNA that interact with different CasX proteins. An exemplary sequence of a scaffold stem loop sequence of a gNA comprises the sequence CCAGCGACUAUGUCGUAUGG (SEQ ID NO: 242). In other embodiments, the disclosure provides gNA variants wherein the scaffold stem loop is replaced with an RNA
stem loop sequence from a heterologous RNA source with proximal 5' and 3' ends, such as, but not limited to stem loop sequences selected from M52, Q(3, Ul hairpin II, Uvsx, or PP7 stem loops. In some cases, the heterologous RNA stem loop of the gNA is capable of binding a protein, an RNA
structure, a DNA sequence, or a small molecule.
d. Extended Stem Loop

[00141] In some embodiments of the gNAs of the disclosure, the scaffold stem loop is followed by the extended stem loop. In some embodiments, the extended stem comprises a synthetic tracr and crRNA fusion that is largely unbound by the CasX protein. In some embodiments, the extended stem loop can be highly malleable. In some embodiments, a single guide gRNA is made with a GAAA tetraloop linker or a GAGAAA linker between the tracr and crRNA in the extended stem loop. In some cases, the targeter and activator of a sgNA are linked to one another by intervening nucleotides and the linker can have a length of from 3 to 20 nucleotides.
In some embodiments of the sgNAs of the disclosure, the extended stem is a large 32-bp loop that sits outside of the CasX protein in the ribonucleoprotein complex. An exemplary sequence of an extended stem loop sequence of a sgNA comprises the sequence GCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGC (SEQ ID NO: 15). In some embodiments, the extended stem loop comprises a GAGAAA spacing sequence. In some embodiments, the disclosure provides gNA variants wherein the extended stem loop is replaced with an RNA stem loop sequence from a heterologous RNA source with proximal 5' and 3' ends, such as, but not limited to stem loop sequences selected from M52, Qf3, Ul hairpin Uvsx, or PP7 stem loops. In such cases, the heterologous RNA stem loop increases the stability of the gNA. In other embodiments, the disclosure provides gNA variants having an extended stem loop region comprising at least 10, at least 100, at least 500, at least 1000, or at least 10,000 nucleotides.
e. Targeting Sequence

[00142] In some embodiments of the gNAs of the disclosure, the extended stem loop is followed by a region that forms part of the triplex, and then the targeting sequence (or "spacer").
The targeting sequence can be designed to target the CasX ribonucleoprotein holo complex to a specific region of the target nucleic acid sequence. Thus, the gNA targeting sequences of the gNAs of the disclosure have sequences complementarity to, and therefore can hybridize to, a portion of the target nucleic acid in a nucleic acid in a eukaryotic cell, (e.g., a eukaryotic chromosome, chromosomal sequence, a eukaryotic RNA, etc.) as a component of the RNP when any one of the PAM sequences TTC, ATC, GTC, or CTC is located 1 nucleotide 5' to the non-target strand sequence complementary to the target sequence.

[00143] In some embodiments, the disclosure provides a gNA wherein the targeting sequence of the gNA is complementary to a target nucleic acid sequence comprising one or more mutations compared to a wild-type gene sequence for purposes of editing the sequence comprising the mutations with the CasX:gNA systems of the disclosure. In some embodiments, the targeting sequence of a gNA is designed to be specific for an exon of the gene of the target nucleic acid. In other embodiments, the targeting sequence of a gNA is designed to be specific for an intron of the gene of the target nucleic acid. In other embodiments, the targeting sequence of the gNA is designed to be specific for an intron-exon junction of the gene of the target nucleic acid. In other embodiments, the targeting sequence of the gNA is designed to be specific for a regulatory element of the gene of the target nucleic acid. In some embodiments, the targeting sequence of the gNA is designed to be complementary to a sequence comprising one or more single nucleotide polymorphisms (SNPs) in a gene of the target nucleic acid.
SNPs that are within the coding sequence or within non-coding sequences are both within the scope of the instant disclosure. In other embodiments, the targeting sequence of the gNA is designed to be complementary to a sequence of an intergenic region of the gene of the target nucleic acid.

[00144] In some embodiments, the targeting sequence of a gNA is designed to be specific for a regulatory element that regulates expression of the gene product of the target nucleic acid. Such regulatory elements include, but are not limited to promoter regions, enhancer regions, intergenic regions, 5' untranslated regions (5' UTR), 3' untranslated regions (3' UTR), conserved elements, and regions comprising cis-regulatory elements. The promoter region is intended to encompass nucleotides within 5 kb of the initiation point of the encoding sequence or, in the case of gene enhancer elements or conserved elements, can be thousands of bp, hundreds of thousands of bp, or even millions of bp away from the encoding sequence of the gene of the target nucleic acid. In some embodiments of the foregoing, the targets are those in which the encoding gene of the target is intended to be knocked out or knocked down such that the encoded protein comprising mutations is not expressed or is expressed at a lower level in a cell.

[00145] In some embodiments, the targeting sequence of a gNA has between 14 and 35 consecutive nucleotides. In some embodiments, the targeting sequence has 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 consecutive nucleotides. In some embodiments, the targeting sequence of the gNA consists of 20 consecutive nucleotides.
In some embodiments, the targeting sequence consists of 19 consecutive nucleotides. In some embodiments, the targeting sequence consists of 18 consecutive nucleotides. In some embodiments, the targeting sequence consists of 17 consecutive nucleotides. In some embodiments, the targeting sequence consists of 16 consecutive nucleotides. In some embodiments, the targeting sequence consists of 15 consecutive nucleotides. In some embodiments, the targeting sequence has 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 consecutive nucleotides and the targeting sequence can comprise 0 to 5, 0 to 4, 0 to 3, or 0 to 2 mismatches relative to the target nucleic acid sequence and retain sufficient binding specificity such that the RNP comprising the gNA comprising the targeting sequence can form a complementary bond with respect to the target nucleic acid.

[00146] In some embodiments, the CasX:gNA system comprises a first gNA and further comprises a second (and optionally a third, fourth, fifth, or more) gNA, wherein the second gNA
or additional gNA has a targeting sequence complementary to a different or overlapping portion of the target nucleic acid sequence compared to the targeting sequence of the first gNA such that multiple points in the target nucleic acid are targeted, and for example, multiple breaks are introduced in the target nucleic acid by the CasX. It will be understood that in such cases, the second or additional gNA is complexed with an additional copy of the CasX
protein. By selection of the targeting sequences of the gNA, defined regions of the target nucleic acid sequence bracketing a mutation can be modified or edited using the CasX:gNA
systems described herein, including facilitating the insertion of a donor template.
f. gNA scaffolds

[00147] With the exception of the targeting sequence region, the remaining regions of the gNA
are referred to herein as the scaffold. In some embodiments, the gNA scaffolds are derived from naturally-occurring sequences, described below as reference gNA. In other embodiments, the gNA scaffolds are variants of reference gNA wherein mutations, insertions, deletions or domain substitutions are introduced to confer desirable properties on the gNA.

[00148] In some embodiments, a reference gRNA comprises a sequence isolated or derived from Deltaproteobacteria. In some embodiments, the sequence is a CasX tracrRNA
sequence.
Exemplary CasX reference tracrRNA sequences isolated or derived from Deltaproteobacteria may include:
ACAUCUGGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUAUGUCGU
AUGGACGAAGCGCUUAUUUAUCGGAGA (SEQ ID NO: 6) and ACAUCUGGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUAUGUCGU
AUGGACGAAGCGCUUAUUUAUCGG (SEQ ID NO: 7). Exemplary crRNA sequences isolated or derived from Deltaproteobacteria may comprise a sequence of CCGAUAAGUAAAACGCAUCAAAG (SEQ ID NO: 243). In some embodiments, a reference gNA comprises a sequence at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89%
identical, at least 89%

identical, at least 90% identical, at least 91% identical, at least 92%
identical, at least 93%
identical, at least 94% identical, at least 95% identical, at least 96%
identical, at least 97%
identical, at least 98% identical, at least 99% identical, at least 99.5%
identical or 100% identical to a sequence isolated or derived from Deltaproteobacteria.

[00149] In some embodiments, a reference guide RNA comprises a sequence isolated or derived from Planctomycetes. In some embodiments, the sequence is a CasX
tracrRNA
sequence. Exemplary reference tracrRNA sequences isolated or derived from Planctomycetes may include:
UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUA
UGGGUAAAGCGCUUAUUUAUCGGAGA (SEQ ID NO: 8) and UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUA
UGGGUAAAGCGCUUAUUUAUCGG (SEQ ID NO: 9). Exemplary crRNA sequences isolated or derived from Planctomycetes may comprise a sequence of UCUCCGAUAAAUAAGAAGCAUCAAAG (SEQ ID NO: 244). In some embodiments, a reference gNA comprises a sequence at least 60% identical, at least 65%
identical, at least 70%
identical, at least 75% identical, at least 80% identical, at least 81%
identical, at least 82%
identical, at least 83% identical, at least 84% identical, at least 85%
identical, at least 86%
identical, at least 86% identical, at least 87% identical, at least 88%
identical, at least 89%
identical, at least 89% identical, at least 90% identical, at least 91%
identical, at least 92%
identical, at least 93% identical, at least 94% identical, at least 95%
identical, at least 96%
identical, at least 97% identical, at least 98% identical, at least 99%
identical, at least 99.5%
identical or 100% identical to a sequence isolated or derived from Planctomycetes.

[00150] In some embodiments, a reference gNA comprises a sequence isolated or derived from Candidatus Sungbacteria. In some embodiments, the sequence is a CasX tracrRNA
sequence.
Exemplary CasX reference tracrRNA sequences isolated or derived from Candidatus Sungbacteria may comprise sequences of: GUUUACACACUCCCUCUCAUAGGGU (SEQ ID
NO: 10), GUUUACACACUCCCUCUCAUGAGGU (SEQ ID NO: 11), UUUUACAUACCCCCUCUCAUGGGAU (SEQ ID NO: 12) and GUUUACACACUCCCUCUCAUGGGGG (SEQ ID NO: 13). In some embodiments, a reference guide RNA comprises a sequence at least 60% identical, at least 65%
identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81%
identical, at least 82%
identical, at least 83% identical, at least 84% identical, at least 85%
identical, at least 86%

identical, at least 86% identical, at least 87% identical, at least 88%
identical, at least 89%
identical, at least 89% identical, at least 90% identical, at least 91%
identical, at least 92%
identical, at least 93% identical, at least 94% identical, at least 95%
identical, at least 96%
identical, at least 97% identical, at least 98% identical, at least 99%
identical, at least 99.5%
identical or 100% identical to a sequence isolated or derived from Candidatus Sungbacteria.

[00151] Table 1 provides the sequences of reference gRNA tracr, cr and scaffold sequences. In some embodiments, the disclosure provides gNA sequences wherein the gNA has a scaffold comprising a sequence having at least one nucleotide modification relative to a reference gNA
sequence having a sequence of any one of SEQ ID NOS: 4-16 of Table 1. It will be understood that in those embodiments wherein a vector comprises a DNA encoding sequence for a gNA, or where a gNA is a gDNA or a chimera of RNA and DNA, that thymine (T) bases can be substituted for the uracil (U) bases of any of the gNA sequence embodiments described herein.
Table 1. Reference gRNA tracr, cr and scaffold sequences SEQ ID Nucleotide Sequence NO.

AUGGACGAAGCGCUUAUUUAUCGGAGAGAAACCGAUAAGUAAAACGCAUCAAAG
UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUA
UGGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG

AUGGACGAAGCGCUUAUUUAUCGGAGA

AUGGACGAAGCGCUUAUUUAUCGG

UGGGUAAAGCGCUUAUUUAUCGGAGA

UGGGUAAAGCGCUUAUUUAUCGG
GUUUACACACUCCCUCUCAUAGGGU

GCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGC

UAAAGCGCUUAUUUAUCGGA
g. gNA Variants

[00152] In another aspect, the disclosure relates to guide nucleic acid variants (referred to herein alternatively as "gNA variant" or "gRNA variant"), which comprise one or more modifications relative to a reference gRNA scaffold. As used herein, "scaffold" refers to all parts to the gNA necessary for gNA function with the exception of the spacer sequence.

[00153] In some embodiments, a gNA variant comprises one or more nucleotide substitutions, insertions, deletions, or swapped or replaced regions relative to a reference gRNA sequence of the disclosure. In some embodiments, a mutation can occur in any region of a reference gRNA
scaffold to produce a gNA variant. In some embodiments, the scaffold of the gNA variant sequence has at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70%, at least 80%, at least 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the sequence of SEQ ID NO: 4 or SEQ ID NO: 5.

[00154] In some embodiments, a gNA variant comprises one or more nucleotide changes within one or more regions of the reference gRNA scaffold that improve a characteristic of the reference gRNA. Exemplary regions include the RNA triplex, the pseudoknot, the scaffold stem loop, and the extended stem loop. In some cases, the variant scaffold stem further comprises a bubble. In other cases, the variant scaffold further comprises a triplex loop region. In still other cases, the variant scaffold further comprises a 5' unstructured region. In some embodiments, the gNA variant scaffold comprises a scaffold stem loop having at least 60%
sequence identity, at least 70% sequence identity, at least 80% sequence identity, at least 90%
sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO:
14. In some embodiments, the gNA variant scaffold comprises a scaffold stem loop having at least 60%
sequence identity to SEQ ID NO: 14. In other embodiments, the gNA variant comprises a scaffold stem loop having the sequence of CCAGCGACUAUGUCGUAGUGG (SEQ ID NO:
245). In other embodiments, the disclosure provides a gNA scaffold comprising, relative to SEQ
ID NO:5, a C18G substitution, a G55 insertion, a Ul deletion, and a modified extended stem loop in which the original 6 nt loop and 13 most-loop-proximal base pairs (32 nucleotides total) are replaced by a Uvsx hairpin (4 nt loop and 5 loop-proximal base pairs; 14 nucleotides total) and the loop-distal base of the extended stem was converted to a fully base-paired stem contiguous with the new Uvsx hairpin by deletion of the A99 and substitution of G65U. In the foregoing embodiment, the gNA scaffold comprises the sequence ACUGGCGCUUTJUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAG

GGAGCAUCAAAG ( SEQ ID NO: 2238).

[00155] All gNA variants that have one or more improved characteristics, or add one or more new functions when the variant gNA is compared to a reference gRNA described herein, are envisaged as within the scope of the disclosure. A representative example of such a gNA variant is guide 174 (SEQ ID NO: 2238), the design of which is described in the Examples. In some embodiments, the gNA variant adds a new function to the RNP comprising the gNA
variant. In some embodiments, the gNA variant has an improved characteristic selected from: improved stability; improved solubility; improved transcription of the gNA; improved resistance to nuclease activity; increased folding rate of the gNA; decreased side product formation during folding; increased productive folding; improved binding affinity to a CasX
protein; improved binding affinity to a target DNA when complexed with a CasX protein; improved gene editing when complexed with a CasX protein; improved specificity of editing when complexed with a CasX protein; and improved ability to utilize a greater spectrum of one or more PAM sequences, including ATC, CTC, GTC, or TTC, in the editing of target DNA when complexed with a CasX
protein, and any combination thereof In some cases, the one or more of the improved characteristics of the gNA variant is at least about 1.1 to about 100,000-fold improved relative to the reference gNA of SEQ ID NO: 4 or SEQ ID NO: 5. In other cases, the one or more improved characteristics of the gNA variant is at least about 1.1, at least about 10, at least about 100, at least about 1000, at least about 10,000, at least about 100,000-fold or more improved relative to the reference gNA of SEQ ID NO: 4 or SEQ ID NO: 5. In other cases, the one or more of the improved characteristics of the gNA variant is about 1.1 to 100,00-fold, about 1.1 to 10,00-fold, about 1.1 to 1,000-fold, about 1.1 to 500-fold, about 1.1 to 100-fold, about 1.1 to 50-fold, about 1.1 to 20-fold, about 10 to 100,00-fold, about 10 to 10,00-fold, about 10 to 1,000-fold, about 10 to 500-fold, about 10 to 100-fold, about 10 to 50-fold, about 10 to 20-fold, about 2 to 70-fold, about 2 to 50-fold, about 2 to 30-fold, about 2 to 20-fold, about 2 to 10-fold, about 5 to 50-fold, about 5 to 30-fold, about 5 to 10-fold, about 100 to 100,00-fold, about 100 to 10,00-fold, about 100 to 1,000-fold, about 100 to 500-fold, about 500 to 100,00-fold, about 500 to 10,00-fold, about 500 to 1,000-fold, about 500 to 750-fold, about 1,000 to 100,00-fold, about 10,000 to 100,00-fold, about 20 to 500-fold, about 20 to 250-fold, about 20 to 200-fold, about 20 to 100-fold, about 20 to 50-fold, about 50 to 10,000-fold, about 50 to 1,000-fold, about 50 to 500-fold, about 50 to 200-fold, or about 50 to 100-fold, improved relative to the reference gNA
of SEQ ID NO: 4 or SEQ ID NO: S. In other cases, the one or more improved characteristics of the gNA variant is about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, 180-fold, 190-fold, 200-fold, 210-fold, 220-fold, 230-fold, 240-fold, 250-fold, 260-fold, 270-fold, 280-fold, 290-fold, 300-fold, 310-fold, 320-fold, 330-fold, 340-fold, 350-fold, 360-fold, 370-fold, 380-fold, 390-fold, 400-fold, 425-fold, 450-fold, 475-fold, or 500-fold improved relative to the reference gNA
of SEQ ID NO: 4 or SEQ ID NO: 5.

[00156] In some embodiments, a gNA variant can be created by subjecting a reference gNA to a one or more mutagenesis methods, such as the mutagenesis methods described herein, below, which may include Deep Mutational Evolution (DME), deep mutational scanning (DMS), error prone PCR, cassette mutagenesis, random mutagenesis, staggered extension PCR, gene shuffling, or domain swapping, in order to generate the gNA variants of the disclosure. The activity of reference gNAs may be used as a benchmark against which the activity of gNA
variants are compared, thereby measuring improvements in function of gNA
variants. In other embodiments, a reference gNA may be subjected to one or more deliberate, targeted mutations, substitutions, or domain swaps in order to produce a gNA variant, for example a rationally designed variant. Exemplary gNA variants produced by such methods are described in the Examples and representative sequences of gNA scaffolds are presented in Table 2.

[00157] In some embodiments, the gNA variant comprises one or more modifications compared to a reference guide nucleic acid scaffold sequence, wherein the one or more modification is selected from: at least one nucleotide substitution in a region of the reference gNA at least one nucleotide deletion in a region of the reference gNA; at least one nucleotide insertion in a region of the reference gNA; a substitution of all or a portion of a region of the reference gNA; a deletion of all or a portion of a region of the reference gNA; or any combination of the foregoing. In some cases, the modification is a substitution of 1 to 15 consecutive or non-consecutive nucleotides in the reference gNA in one or more regions. In other cases, the modification is a deletion of 1 to 10 consecutive or non-consecutive nucleotides in the reference gNA in one or more regions. In other cases, the modification is an insertion of 1 to 10 consecutive or non-consecutive nucleotides in the reference gNA in one or more regions.
In other cases, the modification is a substitution of the scaffold stem loop or the extended stem loop with an RNA stem loop sequence from a heterologous RNA source with proximal 5' and 3' ends. In some cases, a gNA variant of the disclosure comprises two or more modifications in one region relative to a reference gRNA. In other cases, a gNA variant of the disclosure comprises modifications in two or more regions. In other cases, a gNA variant comprises any combination of the foregoing modifications described in this paragraph. In some embodiments, exemplary modifications of gNA of the disclosure include the modifications of Table 24.

[00158] In some embodiments, a 5' G is added to a gNA variant sequence, relative to a reference gRNA, for expression in vivo, as transcription from a U6 promoter is more efficient and more consistent with regard to the start site when the +1 nucleotide is a G. In other embodiments, two 5' Gs are added to generate a gNA variant sequence for in vitro transcription to increase production efficiency, as T7 polymerase strongly prefers a Gin the +1 position and a purine in the +2 position. In some cases, the 5' G bases are added to the reference scaffolds of Table 1. In other cases, the 5' G bases are added to the variant scaffolds of Table 2.

[00159] Table 2 provides exemplary gNA variant scaffold sequences of the disclosure. In Table 2, (-) indicates a deletion at the specified position(s) relative to the reference sequence of SEQ ID NO: 5, (+) indicates an insertion of the specified base(s) at the position indicated relative to SEQ ID NO: 5, (:) indicates the range of bases at the specified start:stop coordinates of a deletion or substitution relative to SEQ ID NO: 5, and multiple insertions, deletions or substitutions are separated by commas; e.g., A14C, T17G. In some embodiments, the gNA
variant scaffold comprises any one of the sequences listed in Table 2, SEQ ID
NOS: 2101-2280, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity thereto.
It will be understood that in those embodiments wherein a vector comprises a DNA encoding sequence for a gNA, or where a gNA is a gDNA or a chimera of RNA and DNA, that thymine (T) bases can be substituted for the uracil (U) bases of any of the gNA sequence embodiments described herein.
Table 2. Exemplary gNA Variant Scaffold Sequences SEQ
NAME or ID NUCLEOTIDE SEQUENCE
Modification NO:
2101 Phage replication UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
stable GGUAAAGCGCAGGUGGGACGACCUCUCGGUCGUCCUAUCUGAAGCAUCAAAG
2102 Kissing loop_bl UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
GGUAAAGCGCUGCUCGACGCGUCCUCGAGCAGAAGCAUCAAAG
2103 Kissing loop_a UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
GGUAAAGCGCUGCUCGCUCCGUUCGAGCAGAAGCAUCAAAG
2104 32, uvsX hairpin GUACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAU

GGGUAAAGCGCCCUCUUCGGAGGGAAGCAUCAAAG

SEQ
NAME or ID NUCLEOTIDE SEQUENCE
Modification NO:
GGUAAAGCGCAGGAGUUUCUAUGGAAACCCUGAAGCAUCAAAG
2106 64, trip mut, GUACUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAU
extended stem GGGUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG
truncation 2107 hyperstable UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
tetraloop GGUAAAGCGCUGCGCUUGCGCAGAAGCAUCAAAG

GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG

GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
2110 CUUCGG loop UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
GGUAAAGCGCUUAUUUAUCGGAGACUUCGGUCCGAUAAAUAAGAAGCAUCAAAG

GGUAAAGCGCACAUGAGGAUUACCCAUGUGAAGCAUCAAAG
2112 -1, A2G, -78, GCUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG

GGUAAAGCGCUGCAUGUCUAAGACAGCAGAAGCAUCAAAG
2114 45,44 hairpin UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG

GGUAAAGCGCAGGGCUUCGGCCGAAGCAUCAAAG
2115 UlA UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
GGUAAAGCGCAAUCCAUUGCACUCCGGAUUGAAGCAUCAAAG
2116 A14C, T17G UACUGGCGCUUUUCUCGCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
2117 CUUCGG loop UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
modified GGUAAAGCGCUUAUUUAUCGGACUUCGGUCCGAUAAAUAAGAAGCAUCAAAG
2118 Kissing 1oop_b2 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
GGUAAAGCGCUGCUCGUUUGCGGCUACGAGCAGAAGCAUCAAAG
2119 -76:78, -83:87 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
GGUAAAGCGCUUAUUUAUCGAGAGAUAAAUAAGAAGCAUCAAAG

GUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
2121 extended stem UACUGGCGCCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAU
truncation GGGUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG

GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
2123 trip mut UACUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
GGUAAAGCGCUUAUUUAUCGGACUUCGGUCCGAUAAAUAAGAAGCAUCAAAG
2124 -76:78 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
GGUAAAGCGCUUAUUUAUCGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
2125 -1:5 GCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUAA
AGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
2126 -83:87 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
GGUAAAGCGCUUAUUUAUCGGAGAGAGAUAAAUAAGAAGCAUCAAAG
2127 =+G28, A82T, - UACUGGCGCUUUUAUCUCAUUACUUUGGAGAGCCAUCACCAGCGACUAUGUCGUAU
84, GGGUAAAGCGCUUAUUUAUCGGAGAGUAUCCGAUAAAUAAGAAGCAUCAAAG
2128 =+51T UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUUCGUAU
GGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
2129 -1:4, +GSA, AGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUA
+G86, AAGCGCUUAUUUAUCGGAGAGAAAUGCCGAUAAAUAAGAAGCAUCAAAG
2130 =+A94 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAAUAAGAAGCAUCAAAG
2131 =+G72 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
GGUAAAGCGCUUAUUGUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG

SEQ
NAME or ID NUCLEOTIDE SEQUENCE
Modification NO:
2132 shorten front, GCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUAA
CUUCGG loop AGCGCUUAUUUAUCGGACUUCGGUCCGAUAAAUAAGCGCAUCAAAG
modified, extend extended GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
2134 -1:3, +G3 GUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGG
UAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
2135 =+C45, +T46 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACCUUAUGUCGUA
UGGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
2136 CUUCGG loop GAUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG
modified, fun GUAAAGCGCUUAUUUAUCGGACUUCGGUCCGAUAAAUAAGAAGCAUCAAAG
start 2137 -93:94 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAAGAAGCAUCAAAG
2138 =+T45 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGAUCUAUGUCGUAU
GGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
2139 -69, -94 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
GGUAAAGGCUUAUUUAUCGGAGAGAAAUCCGAUAAAAAGAAGCAUCAAAG

GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAAAGAAGCAUCAAAG
2141 modified UACUGGCGCUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG
CUUCGG, GUAAAGCGCUUAUUUAUCGGACUUCGGUCCGAUAAAUAAGAAGCAUCAAAG
minus T in 1st triplex 2142 -1:4, +C4, AMC, CGGCGCUUUUCUCGCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGU
T17G, +G72, - AAAGCGCUUAUUGUAUCGAGAGAUAAAUAAGAAGCAUCAAAG
76:78, -83:87 2143 T1C, -73 CACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
GGUAAAGCGCUUAUUUUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
2144 Scaffold uuCG, UACUGGCGCUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUUCGGUCGUAUG
stem uuCG. Stem GGUAAAGCGCUUAUGUAUCGGCUUCGGCCGAUACAUAAGAAGCAUCAAAG
swap, t shorten 2145 Scaffold uuCG, UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUUCGGUCGUAU
stem uuCG. Stem GGGUAAAGCGCUUAUGUAUCGGCUUCGGCCGAUACAUAAGAAGCAUCAAAG
swap 2146 =+G60 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
GGUGAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
2147 no stem Scaffold UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUUCGGUCGUAU
uuCG GGGUAAAG
2148 no stem Scaffold GAUGGGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUUCGGUCGUAUGG
uuCG, fun start GUAAAG
2149 Scaffold uuCG, GAUGGGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUUCGGUCGUAUGG
stem uuCG, fun GUAAAGCGCUUAUUUAUCGGCUUCGGCCGAUAAAUAAGAAGCAUCAAAG
start 2150 Pseudoknots UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
GGUAAAGCGCUACACUGGGAUCGCUGAAUUAGAGAUCGGCGUCCUUUCAUUCUAUA
UACUUUGGAGUUUUAAAAUGUCUCUAAGUACAGAAGCAUCAAAG
2151 Scaffold uuCG, GGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUUCGGUCGUAUGGGU
stem uuCG AAAGCGCUUAUUUAUCGGCUUCGGCCGAUAAAUAAGAAGCAUCAAAG
2152 Scaffold uuCG, GCUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUUCGGUCGUAUG
stem uuCG, no GGUAAAGCGCUUAUUUAUCGGCUUCGGCCGAUAAAUAAGAAGCAUCAAAG
start SEQ
NAME or ID NUCLEOTIDE SEQUENCE
Modification NO:
2153 Scaffold uuCG UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUUCGGUCGUAU

GGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
2154 =+GCTC36 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUGCUCCACCAGCGACUAUGUCG
UAUGGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
2155 G quadriplex UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
telomere basket+ GGUAAAGCGGGGUUAGGGUUAGGGUUAGGGAAGCAUCAAAG
ends 2156 G quadriplex UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
M3q GGUAAAGCGGAGGGAGGGAGGGAGAGGGAAAGCAUCAAAG
2157 G quadriplex UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
telomere basket GGUAAAGCGUUGGGUUAGGGUUAGGGUUAGGGAAAAGCAUCAAAG
no ends 2158 45,44 hairpin UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
(old version) GGUAAAGCGC AGGGCUUCGGCCG GAAGCAUCAAAG
2159 Sarcin-ricin loop UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
GGUAAAGCGCCUGCUCAGUACGAGAGGAACCGCAGGAAGCAUCAAAG
2160 uvsX, C18G UACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
GGUAAAGCGCCCUCUUCGGAGGGAAGCAUCAAAG
2161 truncated stem UACUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
loop, C18G, trip GGUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG
mut (T10C) 2162 short phage rep, UACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG

2163 phage rep loop, UACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG

2164 =+G18, stacked UACUGGCGCCUUUAUCUGCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAU
onto 64 GGGUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG
2165 truncated stem GCUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG
loop, C18G, -1 GUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG

2166 phage rep loop, UACUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
C18G, trip mut GGUAAAGCGCAGGUGGGACGACCUCUCGGUCGUCCUAUCUGAAGCAUCAAAG
(T10C) 2167 short phage rep, UACUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
C18G, trip mut GGUAAAGCGCGGACGACCUCUCGGUCGUCCGAAGCAUCAAAG
(T10C) 2168 uvsX, trip mut UACUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
(T10C) GGUAAAGCGCCCUCUUCGGAGGGAAGCAUCAAAG
2169 truncated stem UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
loop GGUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG
2170 =+A17, stacked UACUGGCGCCUUUAUCAUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAU
onto 64 GGGUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG
2171 3' HDV genomic UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
ribozyme GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAGGGCC
GGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUUCCGAGGGGACCGU
CCCCUCGGUAAUGGCGAAUGGGACCC
2172 phage rep loop, UACUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
trip mut (T10C) GGUAAAGCGCAGGUGGGACGACCUCUCGGUCGUCCUAUCUGAAGCAUCAAAG
2173 -79:80 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
GGUAAAGCGCUUAUUUAUCGGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
2174 short phage rep, UACUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG

trip mut (T10C) GGUAAAGCGCGGACGACCUCUCGGUCGUCCGAAGCAUCAAAG
2175 extra truncated UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG

SEQ
NAME or ID NUCLEOTIDE SEQUENCE
Modification NO:
stem loop GGUAAAGCGCCGGACUUCGGUCCGGAAGCAUCAAAG
2176 T17G, C18G UACUGGCGCUUUUAUCGGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
2177 short phage rep UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
GGUAAAGCGCGGACGACCUCUCGGUCGUCCGAAGCAUCAAAG
2178 uvsX, C18G, -1 GCUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG

2179 uvsx, C18G, trip GCUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG
mut (T10C), -1 GUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG
A2G, HDV -99 2180 3' HDV UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
antigenomic GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAGGGGU
ribozyme CGGCAUGGCAUCUCCACCUCCUCGCGGUCCGACCUGGGCAUCCGAAGGAGGACGCA
CGUCCACUCGGAUGGCUAAGGGAGAGCCA
2181 uvsx, C18G, trip GCUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG
mut (T10C), -1 GUAAAGCGCCCUCUUCGGAGGGCGCAUCAAAG
A2G, HDV
AA(98:99)C
2182 3' HDV ribozyme UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
(Lior Nissim, GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAGUUUU
Timothy Lu) GGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUGCUUCGGCAU
GGCGAAUGGGACCCCGGG
2183 TAC(1:3)GA, GAUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG
stacked onto 64 GUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG
2184 uvsx, -1 A2G GCUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG
GUAAAGCGCCCUCUUCGGAGGGAAGCAUCAAAG
2185 truncated stem GCUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG
loop, C18G, trip GUAAAGCUCUUACGGACUUCGGUCCGUAAGAGCAUCAAAG
mut (T10C), -1 A2G, HDV -99 2186 short phage rep, GCUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG
C18G, trip mut GUAAAGCUCGGACGACCUCUCGGUCGUCCGAGCAUCAAAG
(T10C), -1 A2G, 2187 3' sTRSV WT UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
viral GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAGCCUG
Hammerhead UCACCGGAUGUGCUUUCCGGUCUGAUGAGUCCGUGAGGACGAAACAGG
ribozyme 2188 short phage rep, GCUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG
C18G, -1 A2G GUAAAGCGCGGACGACCUCUCGGUCGUCCGAAGCAUCAAAG
2189 short phage rep, GCUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG
C18G, trip mut GUAAAGCGCGGACGACCUCUCGGUCGUCCGAAGCAUCAAAG
(T10C), -1 A2G, 3' genomic HDV
2190 phage rep loop, GCUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG
C18G, trip mut GUAAAGCUCAGGUGGGACGACCUCUCGGUCGUCCUAUCUGAGCAUCAAAG
(T10C), -1 A2G, 2191 3' HDV ribozyme UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
(Owen Ryan, GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAGGAUG
Jamie Cate) GCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACACCUUCGGGUGGC
GAAUGGGAC

SEQ
NAME or ID NUCLEOTIDE SEQUENCE
Modification NO:
2192 phage rep loop, GCUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG
C18G, -1 A2G GUAAAGCGCAGGUGGGACGACCUCUCGGUCGUCCUAUCUGAAGCAUCAAAG
2193 0.14 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUACU
GGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
2194 -78, G77T UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
GGUAAAGCGCUUAUUUAUCGUGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG

GGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
2196 short phage rep, -GCUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG

2197 truncated stem GCUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG
loop, C18G, trip GUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG
mut (T10C), -1 2198 -1, A2G GCUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG
GUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
2199 truncated stem GCUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG
loop, trip mut GUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG
(T10C), -1 A2G
2200 uvsx, C18G, trip GCUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG
mut (T10C), -1 GUAAAGCGCCCUCUUCGGAGGGAAGCAUCAAAG

2201 phage rep loop, -GCUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG

2202 phage rep loop, GCUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG
trip mut (T10C), GUAAAGCGCAGGUGGGACGACCUCUCGGUCGUCCUAUCUGAAGCAUCAAAG

2203 phage rep loop, GCUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG
C18G, trip mut GUAAAGCGCAGGUGGGACGACCUCUCGGUCGUCCUAUCUGAAGCAUCAAAG
(T10C), -1 A2G
2204 truncated stem UACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
loop, C18G GGUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG
2205 uvsX, trip mut GCUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG
(T10C), -1 A2G GUAAAGCGCCCUCUUCGGAGGGAAGCAUCAAAG
2206 truncated stem GCUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG
loop, -1 A2G GUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG
2207 short phage rep, GCUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG
trip mut (T10C), GUAAAGCGCGGACGACCUCUCGGUCGUCCGAAGCAUCAAAG

2208 5'HDV ribozyme GAUGGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACACCUUCGGG
(Owen Ryan, UGGCGAAUGGGACUACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCG
Jamie Cate) ACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAA
GCAUCAAAG
2209 511-1DV genomic GGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUUCCGAGGGGA
ribozyme CCGUCCCCUCGGUAAUGGCGAAUGGGACCCUACUGGCGCUUUUAUCUCAUUACUUU
GAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGAGAGA
AAUCCGAUAAAUAAGAAGCAUCAAAG
2210 truncated stem GCUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG
loop, C18G, trip GUAAAGCGCUUACGGACUUCGGUCCGUAAGCGCAUCAAAG
mut (T10C), -1 A2G, HDV
AA(98:99)C
2211 5'env25 pistol CGUGGUUAGGGCCACGUUAAAUAGUUGCUUAAGCCCUAAGCGUUGAUCUUCGGAUC
ribozyme (with AGGUGCAAUACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAU

SEQ
NAME or ID NUCLEOTIDE SEQUENCE
Modification NO:
an added GUCGUAUGGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUC
CUUCGG loop) AAAG

antigeno mic CGCACGUCCACUCGGAUGGCUAAGGGAGAGCCAUACUGGCGCUUUUAUCUCAUUAC
ribozyme UUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGAG
AGAAAUCCGAUAAAUAAGAAGCAUCAAAG
2213 3' Hammerhead UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
ribozyme (Lior GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAGCCAG
Nissim, Timothy UACUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCUACUGGCGCUUUUAUCU
Lu) guide CAU
scaffold scar 2214 =+A27, stacked UACUGGCGCCUUUAUCUCAUUACUUUAGAGAGCCAUCACCAGCGACUAUGUCGUAU
onto 64 GGGUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG
2215 51-lammerhead CGACUACUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCUAGUCGUACUGGC
ribozyme (Lior GCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUAAAG
Nissim, Timothy CGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
Lu) smaller scar 2216 Phage rep loop, GCUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG
C18G, trip mut GUAAAGCGCAGGUGGGACGACCUCUCGGUCGUCCUAUCUGCGCAUCAAAG
(T10C), -1 A2G, HDV
AA(98 :99)C
2217 -27, stacked onto UACUGGCGCCUUUAUCUCAUUACUUUAGAGCCAUCACCAGCGACUAUGUCGUAUGG

2218 3' Hatchet UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAGCAUU
CCUCAGAAAAUGACAAACCUGUGGGGCGUAAGUAGAUCUUCGGAUCUAUGAUCGUG
CAGACGUUAAAAUCAGGU
2219 3' Hammerhead UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
ribozyme (Lior GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAGCGAC
Nissim, Timothy UACUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCUAGUCGCGUGUAGCGAA
Lu) GCA
2220 5'Hatchet CAUUCCUCAGAAAAUGACAAACCUGUGGGGCGUAAGUAGAUCUUCGGAUCUAUGAU
CGUGCAGACGUUAAAAUCAGGUUACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCA
UCACCAGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAU
AAAUAAGAAGCAUCAAAG
2221 511-1DV ribozyme UUUUGGCCGGCAUGGUCCCAGCCUCCUCGCUGGCGCCGGCUGGGCAACAUGCUUCG
(Lior Nissim, GCAUGGCGAAUGGGACCCCGGGUACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCA
Timothy Lu) UCACCAGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAU
AAAUAAGAAGCAUCAAAG
2222 51-lammerhead CGACUACUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCUAGUCGCGUGUAG
ribozyme (Lior CGAAGCAUACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUG
Nissim, Timothy TiCGUAUGGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCA
Lu) AAG
2223 3' HH15 Minimal UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
Hammerhead GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAGGGGA
ribozyme GCCCCGCUGAUGAGGUCGGGGAGACCGAAAGGGACUUCGGUCCCUACGGGGCUCCC
2224 5' RBMX CCACCCCCACCACCACCCCCACCCCCACCACCACCCUACUGGCGCUUUUAUCUCAU
recruiting motif UACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCG
GAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
2225 3' Hammerhead UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
ribozyme (Lior GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAGCGAC
Nissim, Timothy UACUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCUAGUCG
Lu) smaller scar SEQ
NAME or ID NUCLEOTIDE SEQUENCE
Modification NO:
2226 3' env25 pistol UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
ribozyme (with GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAGCGUG
an added GUUAGGGCCACGUUAAAUAGUUGCUUAAGCCCUAAGCGUUGAUCUUCGGAUCAGGU
CUUCGG loop) GCAA
2227 3' Env-9 Twister UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG

GGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAGGGCA
AUAAAGCGGUUACAAGCCCGCAAAAAUAGCAGAGUAAUGUCGCGAUAGCGCGGCAU
UAAUGCAGCUUUAUUG
2228 = A _TT_ _T _ A C
+_ TCA UACUGGCGCUUUUAUCUCAUUACUAUUAUCUCAUUACUUUGAGAGCCAUCACCAGC

AGCAUCAAAG
2229 5'Env-9 Twister GGCAAUAAAGCGGUUACAAGCCCGCAAAAAUAGCAGAGUAAUGUCGCGAUAGCGCG
GCAUUAAUGCAGCUUUAUUGUACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUC
ACCAGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAA
AUAAGAAGCAUCAAAG
2230 3' Twisted Sister UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG

GCAAGGCCGACGGCAUCCGCCGCCGCUGGUGCAAGUCCAGCCGCCCCUUCGGGGGC
GGGCGCUCAUGGGUAAC
2231 no stem UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG
GGUAAAG
2232 511-1H15 Minimal GGGAGCCCCGCUGAUGAGGUCGGGGAGACCGAAAGGGACUUCGGUCCCUACGGGGC
Hammerhead UCCCUACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCG
ribozyme UAUGGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
2233 51Hammerhead CCAGUACUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUCUACUGGCGCUUUU
ribozyme (Lior AUCUCAUUACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUG
Nissim, Timothy uCGUAUGGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCA
Lu) guide AAG
scaffold scar 2234 5'Twisted Sister ACCCGCAAGGCCGACGGCAUCCGCCGCCGCUGGUGCAAGUCCAGCCGCCCCUUCGG

CACCAGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGAGAGAAAUCCGAUA
AAUAAGAAGCAUCAAAG
2235 5'sTRSV WT CCUGUCACCGGAUGUGCUUUCCGGUCUGAUGAGUCCGUGAGGACGAAACAGGUACU
viral GGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUA
Hammerhead AAGCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGCAUCAAAG
ribozyme 2236 148, =+G55, GUACUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG
stacked onto 64 UGGGUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG
2237 158, GUACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG
103+148(+G55) - UGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG
99, G65U
2238 174, Uvsx ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUG
Extended stem GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG
with [A99]
G65U), Cl8G,AG55, [GT-1]
2239 175, extended ACUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG
stem truncation, GUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG
T10C, [GT-1]
2240 176, 174 with GCUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUG
Al G substitution GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG
for T7 SEQ
NAME or ID NUCLEOTIDE SEQUENCE
Modification NO:
transcription 2241 177, 174 with ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG
bubble (+G55) GUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG
removed 2242 181, stem 42 (truncated stem loop);
T10C,C18G,[GT ACUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG
-11 (95+[GT-1]) GUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG
2243 182, stem 42 (truncated stem loop); ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG
C18GJGT-1] GUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG
2244 183, stem 42 (truncated stem loop);
C18G,AG55,[GT- ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUG

2245 184, stem 48 (uvsx, -99 g65t);
C18G,AT55,[GT- ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUUG

2246 185, stem 42 (truncated stem loop);
C18G,AT55,[GT- ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUUG

2247 186, stem 42 (truncated stem loop);
T10C,AA17,[GT- ACUGGCGCCUUUAUCAUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUG

2248 187, stem 46 (uvsx);
C18G,AG55,[GT- ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUG

2249 188, stem 50 (ms2 U15C, -99, g65t);
C18G,AG55,[GT- ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUG

2250 189, 174 + ACUGGCACUUUUACCUGAUUACUUUGAGAGCCAACACCAGCGACUAUGUCGUAGUG
G8A;T15C;T35A GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG

190, 174 + G8A GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG

191, 174 + G8C GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG

192, 174 + T15C GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG

193, 174 + T35A GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG
2255 195, 175 + C18G
ACUGGCACCUUUACCUGAUUACUUUGAGAGCCAACACCAGCGACUAUGUCGUAUGG
G8A;T15C;T35A GUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG

SEQ
NAME or ID NUCLEOTIDE SEQUENCE
Modification NO:
2256 196, 175 + C18G ACUGGCACCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG
+ G8A GUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG
2257 197, 175 + C18G ACUGGCCCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGG
+ G8C GUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG
2258 198, 175 + C18G ACUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAACACCAGCGACUAUGUCGUAUGG
+ T35A GUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG
2259 199, 174 + A2G
(test G
transcription at GCUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUG
start; ccGCT...) GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG
2260 200, 174 + AG1 GACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGU
(ccGACT...) GGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG
2261 201, 174 + ACUGGCGCCUUUAUCUGAUUACUUUGGAGAGCCAUCACCAGCGACUAUGUCGUAGU
T10C;AG28 GGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG
2262 202, 174 + ACUGGCGCAUUUAUCUGAUUACUUUGUGAGCCAUCACCAGCGACUAUGUCGUAGUG
T10A;A28T GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG

203, 174 + T10C GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG

204, 174 + AG28 GGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG

205, 174 + T10A GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG

206, 174 + A28T GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG

207, 174 + AT15 GGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG

208, 174 + [T4] GUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG

209, 174 + C16A GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG

210, 174 + AT17 GGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG
2271 211, 174 + T35G
(compare with 174 + T35A ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAGCACCAGCGACUAUGUCGUAGUG
above) GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG
2272 212, 174 +UllG, A105G (A86G), ACUGGCGCUGUUAUCUGAUUACUUCGAGAGCCAUCACCAGCGACUAUGUCGUAGUG

2273 213, 174 +U11C, A105G (A86G), ACUGGCGCUCUUAUCUGAUUACUUCGAGAGCCAUCACCAGCGACUAUGUCGUAGUG

2274 214, 174+U12G;
A106G (A87G), ACUGGCGCUUGUAUCUGAUUACUCUGAGAGCCAUCACCAGCGACUAUGUCGUAGUG

2275 215, 174+U12C;
A106G (A87G), ACUGGCGCUUCUAUCUGAUUACUCUGAGAGCCAUCACCAGCGACUAUGUCGUAGUG

2276 216, 174_tx_11.G,87. ACUGGCGCUUUGAUCUGAUUACCUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUG
G,22.0 GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAGG
2277 217, ACUGGCGCUUUCAUCUGAUUACCUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUG
174_tx_11.C,87. GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAGG

SEQ
NAME or ID NUCLEOTIDE SEQUENCE
Modification NO:
G,22.0 218, 174 +UllG GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG
2279 219, 174 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUG
+A105G (A86G) GGUAAAGCUCCCUCUUCGGAGGGAGCAUCGAAG

220, 174 +U26C GGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG

[00160] In some embodiments, the gNA variant comprises a tracrRNA stem loop comprising the sequence -UUU-N4-25-UUU- (SEQ ID NO: 240). For example, the gNA variant comprises a scaffold stem loop or a replacement thereof, flanked by two triplet U motifs that contribute to the triplex region. In some embodiments, the scaffold stem loop or replacement thereof comprises at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, at least 23 nucleotides, at least 24 nucleotides, or at least 25 nucleotides.

[00161] In some embodiments, the gNA variant comprises a crRNA sequence with -AAAG- in a location 5' to the spacer region. In some embodiments, the -AAAG- sequence is immediately 5' to the spacer region.

[00162] In some embodiments, the at least one nucleotide modification to a reference gNA to produce a gNA variant comprises at least one nucleotide deletion in the CasX
variant gNA
relative to the reference gRNA. In some embodiments, a gNA variant comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 consecutive or non-consecutive nucleotides relative to a reference gNA. In some embodiments, the at least one deletion comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more consecutive nucleotides relative to a reference gNA. In some embodiments, the gNA variant comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more nucleotide deletions relative to the reference gNA, and the deletions are not in consecutive nucleotides. In those embodiments where there are two or more non-consecutive deletions in the gNA variant relative to the reference gRNA, any length of deletions, and any combination of lengths of deletions, as described herein, are contemplated as within the scope of the disclosure. For example, in some embodiments, a gNA variant may comprise a first deletion of one nucleotide, and a second deletion of two nucleotides and the two deletions are not consecutive. In some embodiments, a gNA variant comprises at least two deletions in different regions of the reference gRNA. In some embodiments, a gNA variant comprises at least two deletions in the same region of the reference gRNA. For example, the regions may be the extended stem loop, scaffold stem loop, scaffold stem bubble, triplex loop, pseudoknot, triplex, or a 5' end of the gNA variant. The deletion of any nucleotide in a reference gRNA is contemplated as within the scope of the disclosure.

[00163] In some embodiments, the at least one nucleotide modification of a reference gRNA to generate a gNA variant comprises at least one nucleotide insertion. In some embodiments, a gNA variant comprises an insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive or non-consecutive nucleotides relative to a reference gRNA. In some embodiments, the at least one nucleotide insertion comprises an insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more consecutive nucleotides relative to a reference gRNA.
In some embodiments, the gNA variant comprises 2 or more insertions relative to the reference gRNA, and the insertions are not consecutive. In those embodiments where there are two or more non-consecutive insertions in the gNA variant relative to the reference gRNA, any length of insertions, and any combination of lengths of insertions, as described herein, are contemplated as within the scope of the disclosure. For example, in some embodiments, a gNA
variant may comprise a first insertion of one nucleotide, and a second insertion of two nucleotides and the two insertions are not consecutive. In some embodiments, a gNA variant comprises at least two insertions in different regions of the reference gRNA. In some embodiments, a gNA variant comprises at least two insertions in the same region of the reference gRNA.
For example, the regions may be the extended stem loop, scaffold stem loop, scaffold stem bubble, triplex loop, pseudoknot, triplex, or a 5' end of the gNA variant. Any insertion of A, G, C, U (or T, in the corresponding DNA) or combinations thereof at any location in the reference gRNA is contemplated as within the scope of the disclosure.

[00164] In some embodiments, the at least one nucleotide modification of a reference gRNA to genereate a gNA variant comprises at least one nucleic acid substitution. In some embodiments, a gNA variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more consecutive or non-consecutive substituted nucleotides relative to a reference gRNA. In some embodiments, a gNA variant comprises 1-4 nucleotide substitutions relative to a reference gRNA. In some embodiments, the at least one substitution comprises a substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more consecutive nucleotides relative to a reference gRNA. In some embodiments, the gNA variant comprises 2 or more substitutions relative to the reference gRNA, and the substitutions are not consecutive. In those embodiments where there are two or more non-consecutive substitutions in the gNA variant relative to the reference gRNA, any length of substituted nucleotides, and any combination of lengths of substituted nucleotides, as described herein, are contemplated as within the scope of the disclosure. For example, in some embodiments, a gNA variant may comprise a first substitution of one nucleotide, and a second substitution of two nucleotides and the two substitutions are not consecutive. In some embodiments, a gNA variant comprises at least two substitutions in different regions of the reference gRNA. In some embodiments, a gNA variant comprises at least two substitutions in the same region of the reference gRNA. For example, the regions may be the triplex, the extended stem loop, scaffold stem loop, scaffold stem bubble, triplex loop, pseudoknot, triplex, or a 5' end of the gNA variant. Any substitution of A, G, C, U (or T, in the corresponding DNA) or combinations thereof at any location in the reference gRNA is contemplated as within the scope of the disclosure.

[00165] Any of the substitutions, insertions and deletions described herein can be combined to generate a gNA variant of the disclosure. For example, a gNA variant can comprise at least one substitution and at least one deletion relative to a reference gRNA, at least one substitution and at least one insertion relative to a reference gRNA, at least one insertion and at least one deletion relative to a reference gRNA, or at least one substitution, one insertion and one deletion relative to a reference gRNA.

[00166] In some embodiments, the gNA variant comprises a scaffold region at least 20%
identical, at least 30% identical, at least 40% identical, at least 50%
identical, at least 60%
identical, at least 65% identical, at least 70% identical, at least 75%
identical, at least 80%
identical, at least 85% identical, at least 90% identical, at least 91%
identical, at least 92%
identical, at least 93% identical, at least 94% identical, at least 95%
identical, at least 96%
identical, at least 97% identical, at least 98% identical, or at least 99%
identical to any one of SEQ ID NOS: 4-16. In some embodiments, the gNA variant comprises a scaffold region at least 60% homologous (or identical) to any one of SEQ ID NOS: 4-16.

[00167] In some embodiments, the gNA variant comprises a tracr stem loop at least 60%
identical, at least 65% identical, at least 70% identical, at least 75%
identical, at least 80%
identical, at least 85% identical, at least 90% identical, at least 91%
identical, at least 92%

identical, at least 93% identical, at least 94% identical, at least 95%
identical, at least 96%
identical, at least 97% identical, at least 98% identical, or at least 99%
identical to SEQ ID NO:
14. In some embodiments, the gNA variant comprises a tracr stem loop at least 60%
homologous (or identical) to SEQ ID NO: 14.

[00168] In some embodiments, the gNA variant comprises an extended stem loop at least 60%
identical, at least 65% identical, at least 70% identical, at least 75%
identical, at least 80%
identical, at least 85% identical, at least 90% identical, at least 91%
identical, at least 92%
identical, at least 93% identical, at least 94% identical, at least 95%
identical, at least 96%
identical, at least 97% identical, at least 98% identical, or at least 99%
identical to SEQ ID NO:
15. In some embodiments, the gNA variant comprises an extended stem loop at least 60%
homologous (or identical) to SEQ ID NO: 15.

[00169] In some embodiments, a gNA variant comprises a sequence of any one of SEQ ID
NOs: 412-3295. In some embodiments, a gNA variant comprises a sequence of any one of SEQ
ID NOS: 2236, 2237, 2238, 2241, 2244, 2248, 2249, or 2259-2280. In some embodiments, a gNA variant comprises a sequence of any one of SEQ ID NOS: 2236, 2237, 2238, 2241, 2244, 2248, 2249, or 2259-2280.

[00170] In some embodiments, the gNA variant comprises an exogenous extended stem loop, with such differences from a reference gNA described as follows. In some embodiments, an exogenous extended stem loop has little or no identity to the reference stem loop regions disclosed herein (e.g., SEQ ID NO: 15). In some embodiments, an exogenous stem loop is at least 10 bp, at least 20 bp, at least 30 bp, at least 40 bp, at least 50 bp, at least 60 bp, at least 70 bp, at least 80 bp, at least 90 bp, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp, at least 900 bp, at least 1,000 bp, at least 2,000 bp, at least 3,000 bp, at least 4,000 bp, at least 5,000 bp, at least 6,000 bp, at least 7,000 bp, at least 8,000 bp, at least 9,000 bp, at least 10,000 bp, at least 12,000 bp, at least 15,000 bp or at least 20,000 bp. In some embodiments, the gNA variant comprises an extended stem loop region comprising at least 10, at least 100, at least 500, at least 1000, or at least 10,000 nucleotides. In some embodiments, the heterologous stem loop increases the stability of the gNA. In some embodiments, the heterologous RNA stem loop is capable of binding a protein, an RNA structure, a DNA sequence, or a small molecule. In some embodiments, an exogenous stem loop region comprises an RNA stem loop or hairpin, for example a thermostable RNA such as M52 (ACAUGAGGAUUACCCAUGU; SEQ ID NO: 4278), Qf3 (UGCAUGUCUAAGACAGCA; SEQ ID NO: 4279), Ul hairpin II
(AAUCCAUUGCACUCCGGAUU; SEQ ID NO:4280), Uvsx (CCUCUUCGGAGG; SEQ ID
NO: 4281), PP7 (AGGAGUUUCUAUGGAAACCCU; SEQ ID NO: 4282), Phage replication loop (AGGUGGGACGACCUCUCGGUCGUCCUAUCU; SEQ ID NO: 4283), Kissingloop a (UGCUCGCUCCGUUCGAGCA; SEQ ID NO: 4284), Kissing loop bl (UGCUCGACGCGUCCUCGAGCA; SEQ ID NO: 4285), Kissing loop b2 (UGCUCGUUUGCGGCUACGAGCA; SEQ ID NO: 4286), G quadriplex M3q (AGGGAGGGAGGGAGAGG; SEQ ID NO: 4287), G quadriplex telomere basket (GGUUAGGGUUAGGGUUAGG; SEQ ID NO: 4288), Sarcin-ricin loop (CUGCUCAGUACGAGAGGAACCGCAG; SEQ ID NO: 4289) or Pseudoknots (UACACUGGGAUCGCUGAAUUAGAGAUCGGCGUCCUUUCAUUCUAUAUACUUUGG
AGUUUUAAAAUGUCUCUAAGUACA; SEQ ID NO: 4290). In some embodiments, an exogenous stem loop comprises an RNA scaffold. As used herein, an "RNA
scaffold" refers to a multi-dimensional RNA structure capable of interacting with and organizing or localizing one or more proteins. In some embodiments, the RNA scaffold is synthetic or non-naturally occurring.
In some embodiments, an exogenous stem loop comprises a long non-coding RNA
(lncRNA).
As used herein, a lncRNA refers to a non-coding RNA that is longer than approximately 200 bp in length. In some embodiments, the 5' and 3' ends of the exogenous stem loop are base paired, i.e., interact to form a region of duplex RNA. In some embodiments, the 5' and 3' ends of the exogenous stem loop are base paired, and one or more regions between the 5' and 3' ends of the exogenous stem loop are not base paired. In some embodiments, the at least one nucleotide modification comprises: (a) substitution of 1 to 15 consecutive or non-consecutive nucleotides in the gNA variant in one or more regions; (b) a deletion of 1 to 10 consecutive or non-consecutive nucleotides in the gNA variant in one or more regions; (c) an insertion of 1 to 10 consecutive or non-consecutive nucleotides in the gNA variant in one or more regions; (d) a substitution of the scaffold stem loop or the extended stem loop with an RNA stem loop sequence from a heterologous RNA source with proximal 5' and 3' ends; or any combination of (a)-(d).

[00171] In some embodiments, a gNA variant comprises a sequence or subsequence of any one of SEQ ID NOs: 412-3295 and an a sequence of an exogenous stem loop. In some embodiments, a gNA variant comprises a sequence or subsequence of any one of SEQ ID NOS:
2236, 2237, 2238, 2241, 2244, 2248, 2249, or 2259-2280 and a sequence of an exogenous stem loop. In some embodiments, a gNA variant comprises a sequence or subsequence of any one of SEQ ID

NOS: 2236, 2237, 2238, 2241, 2244, 2248, 2249, or 2259-2280 and a sequence of an exogenous stem loop.

[00172] In some embodiments, the gNA variant comprises a scaffold stem loop having at least 60% identity to SEQ ID NO: 14. In some embodiments, the gNA variant comprises a scaffold stem loop having at least 60% identity, at least 70% identity, at least 80%
identity, at least 90%
identity, at least 95% identity, at least 98% identity or at least 99%
identity to SEQ ID NO: 14.
In some embodiments, the gNA variant comprises a scaffold stem loop comprising SEQ ID NO:
14.

[00173] In some embodiments, the gNA variant comprises a scaffold stem loop sequence of CCAGCGACUAUGUCGUAGUGG (SEQ ID NO: 245). In some embodiments, the gNA
variant comprises a scaffold stem loop sequence of CCAGCGACUAUGUCGUAGUGG (SEQ
ID NO: 245) with at least 1, 2, 3, 4, or 5 mismatches thereto.

[00174] In some embodiments, the gNA variant comprises an extended stem loop region comprising less than 32 nucleotides, less than 31 nucleotides, less than 30 nucleotides, less than 29 nucleotides, less than 28 nucleotides, less than 27 nucleotides, less than 26 nucleotides, less than 25 nucleotides, less than 24 nucleotides, less than 23 nucleotides, less than 22 nucleotides, less than 21 nucleotides, or less than 20 nucleotides. In some embodiments, the gNA variant comprises an extended stem loop region comprising less than 32 nucleotides. In some embodiments, the gNA variant further comprises a thermostable stem loop.

[00175] In some embodiments, a sgRNA variant comprises a sequence of SEQ ID
NO: 2104, 2106, SEQ ID NO: 2163, SEQ ID NO: 2107, SEQ ID NO: 2164, SEQ ID NO: 2165, SEQ
ID
NO: 2166, SEQ ID NO: 2103, SEQ ID NO: 2167, SEQ ID NO: 2105, SEQ ID NO: 2108, SEQ
ID NO: 2112, SEQ ID NO: 2160, SEQ ID NO: 2170, SEQ ID NO: 2114, SEQ ID NO:
2171, SEQ ID NO: 2112, SEQ ID NO: 2173, SEQ ID NO: 2102, SEQ ID NO: 2174, SEQ ID NO:

2175, SEQ ID NO: 2109, SEQ ID NO: 2176, SEQ ID NO: 2238, SEQ ID NO: 2239, SEQ
ID
NO: 2240, or SEQ ID NO: 2241.

[00176] In some embodiments, the gNA variant comprises one or more additional changes to a sequence of any one of SEQ ID NOs: 2201-2280. In some embodiments, the gNA
variant comprises a sequence of any one of SEQ ID NOS: 2236, 2237, 2238, 2241, 2244, 2248, 2249, or 2259-2280, or having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity thereto. In some embodiments, the gNA variant comprises one or more additional changes to a sequence of any one of SEQ ID NOs: 2201-2280. In some embodiments, the gNA variant comprises the sequence of any one of SEQ ID NOS: 2236, 2237, 2238, 2241, 2244, 2248, 2249, or 2259-2280.

[00177] In some embodiments, a sgRNA variant comprises one or more additional changes to a sequence of SEQ ID NO: 2104, SEQ ID NO: 2163, SEQ ID NO: 2107, SEQ ID NO:
2164, SEQ
ID NO: 2165, SEQ ID NO: 2166, SEQ ID NO: 2103, SEQ ID NO: 2167, SEQ ID NO:
2105, SEQ ID NO: 2108, SEQ ID NO: 2112, SEQ ID NO: 2160, SEQ ID NO: 2170, SEQ ID NO:

2114, SEQ ID NO: 2171, SEQ ID NO: 2112, SEQ ID NO: 2173, SEQ ID NO: 2102, SEQ
ID
NO: 2174, SEQ ID NO: 2175, SEQ ID NO: 2109, SEQ ID NO: 2176, SEQ ID NO: 2238, SEQ
ID NO: 2239, SEQ ID NO: 2240, or SEQ ID NO: 2241.

[00178] In some embodiments of the gNA variants of the disclosure, the gNA
variant comprises at least one modification, wherein the at least one modification compared to the reference guide scaffold of SEQ ID NO: 5 is selected from one or more of: (a) a C18G
substitution in the triplex loop; (b) a G55 insertion in the stem bubble; (c) a Ul deletion; (d) a modification of the extended stem loop wherein (i) a 6 nt loop and 13 loop-proximal base pairs are replaced by a Uvsx hairpin; and (ii) a deletion of A99 and a substitution of G65U that results in a loop-distal base that is fully base-paired. In such embodiments, the gNA
variant comprises the sequence of any one of SEQ ID NOS: 2236, 2237, 2238, 2241, 2244, 2248, 2249, or 2259-2280.

[00179] In some embodiments, the scaffold of the gNA variant comprises the sequence of any one of SEQ ID NOS: 2201-2280 of Table 2. In some embodiments, the scaffold of the gNA
consists or consists essentially of the sequence of any one of SEQ ID NOS:
2201-2280. In some embodiments, the scaffold of the gNA variant sequence is at least about 60%
identical, at least about 65% identical, at least about 70% identical, at least about 75%
identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91%
identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97%
identical, at least about 98% identical or at least about 99% identical to any one of SEQ ID NOS:
2201 to 2280.

[00180] In some embodiments, the gNA variant further comprises a spacer (or targeting sequence) region, described more fully, supra, which comprises at least 14 to about 35 nucleotides wherein the spacer is designed with a sequence that is complementary to a target DNA. In some embodiments, the gNA variant comprises a targeting sequence of at least 10 to 30 nucleotides complementary to a target DNA. In some embodiments, the targeting sequence has 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides.
In some embodiments, the gNA variant comprises a targeting sequence having 20 nucleotides. In some embodiments, the targeting sequence has 25 nucleotides. In some embodiments, the targeting sequence has 24 nucleotides. In some embodiments, the targeting sequence has 23 nucleotides. In some embodiments, the targeting sequence has 22 nucleotides.
In some embodiments, the targeting sequence has 21 nucleotides. In some embodiments, the targeting sequence has 20 nucleotides. In some embodiments, the targeting sequence has 19 nucleotides.
In some embodiments, the targeting sequence has 18 nucleotides. In some embodiments, the targeting sequence has 17 nucleotides. In some embodiments, the targeting sequence has 16 nucleotides. In some embodiments, the targeting sequence has 15 nucleotides.
In some embodiments, the targeting sequence has 14 nucleotides.

[00181] In some embodiments, the scaffold of the gNA variant is a variant comprising one or more additional changes to a sequence of a reference gRNA that comprises SEQ
ID NO: 4 or SEQ ID NO: 5. In those embodiments where the scaffold of the reference gRNA is derived from SEQ ID NO: 4 or SEQ ID NO: 5, the one or more improved or added characteristics of the gNA
variant are improved compared to the same characteristic in SEQ ID NO: 4 or SEQ ID NO: 5.

[00182] In some embodiments, the scaffold of the gNA variant is part of an RNP
with a reference CasX protein comprising SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.
In other embodiments, the scaffold of the gNA variant is part of an RNP with a CasX
variant protein comprising any one of the sequences of Tables 3, 8, 9, 10 and 12, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In the foregoing embodiments, the gNA further comprises a spacer sequence.
h. Chemically Modified gNAs

[00183] In some embodiments, the disclosure provides chemically-modified gNAs.
In some embodiments, the present disclosure provides a chemically-modified gNA that has guide NA
functionality and has reduced susceptibility to cleavage by a nuclease. A gNA
that comprises any nucleotide other than the four canonical ribonucleotides A, C, G, and U, or a deoxynucleotide, is a chemically modified gNA. In some cases, a chemically-modified gNA
comprises any backbone or internucleotide linkage other than a natural phosphodiester internucleotide linkage. In certain embodiments, the retained functionality includes the ability of the modified gNA to bind to a CasX of any of the embodiments described herein.
In certain embodiments, the retained functionality includes the ability of the modified gNA to bind to a target nucleic acid sequence. In certain embodiments, the retained functionality includes targeting a CasX protein or the ability of a pre-complexed RNP to bind to a target nucleic acid sequence. In certain embodiments, the retained functionality includes the ability to nick a target polynucleotide by a CasX-gNA. In certain embodiments, the retained functionality includes the ability to cleave a target nucleic acid sequence by a CasX-gNA. In certain embodiments, the retained functionality is any other known function of a gNA in a recombinant system with a CasX chimera protein of the embodiments of the disclosure.

[00184] In some embodiments, the disclosure provides a chemically-modified gNA
in which a nucleotide sugar modification is incorporated into the gNA selected from the group consisting of 2'-0¨C1.4alkyl such as 2'-0-methyl (2'-0Me), 2'-deoxy (2'-H), 2'-0¨C1.3alkyl-O¨C1.3alkyl such as 2'-methoxyethyl ("2'-MOE"), 2'-fluoro ("2'-F"), 2'-amino ("2'-NH2"), 2'-arabinosyl ("2'-arabino") nucleotide, 2'-F-arabinosyl ("2'-F-arabino") nucleotide, 2'-locked nucleic acid ("LNA") nucleotide, 2'-unlocked nucleic acid ("ULNA") nucleotide, a sugar in L
form ("L-sugar"), and 4'-thioribosyl nucleotide. In other embodiments, an internucleotide linkage modification incorporated into the guide RNA is selected from the group consisting of:
phosphorothioate "P(S)" (P(S)), phosphonocarboxylate (P(CH2).COOR) such as phosphonoacetate "PACE" (P(CH2C00-)), thiophosphonocarboxylate ((S)P(CH2).COOR) such as thiophosphonoacetate "thioPACE" ((S)P(CH2).000-)), alkylphosphonate (P(C,.,alkyl) such as methylphosphonate ¨P(CH,), boranophosphonate (P(BH,)), and phosphorodithioate (P(S)2).

[00185] In certain embodiments, the disclosure provides a chemically-modified gNA in which a nucleobase ("base") modification is incorporated into the gNA selected from the group consisting of: 2-thiouracil ("2-thioU"), 2-thiocytosine ("2-thioC"), 4-thiouracil ("4-thioU"), 6-thioguanine ("6-thioG"), 2-aminoadenine ("2-aminoA"), 2-aminopurine, pseudouracil, hypoxanthine, 7-deazaguanine, 7-deaza-8-azaguanine, 7-deazaadenine, 7-deaza-8-azaadenine, 5-methylcytosine ("5-methylC"), 5-methyluracil ("5-methylU"), 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5,6-dehydrouracil, 5-propynylcytosine, 5-propynyluracil, ethynylcytosine, 5-ethynyluracil, 5-allyluracil ("5-ally1U"), 5-allylcytosine ("5-ally1C"), 5-aminoallyluracil ("5-aminoally1U"), 5-aminoallyl-cytosine ("5-aminoally1C"), an abasic nucleotide, Z base, P base, Unstructured Nucleic Acid ("UNA"), isoguanine ("isoG"), isocytosine ("isoC"), 5-methyl-2-pyrimidine, x(A,G,C,T) and y(A,G,C,T).

[00186] In other embodiments, the disclosure provides a chemically-modified gNA in which one or more isotopic modifications are introduced on the nucleotide sugar, the nucleobase, the phosphodiester linkage and/or the nucleotide phosphates, including nucleotides comprising one or more 15N, 13,,, 14C, deuterium, 3H, 32p, 125T1 , 131j atoms or other atoms or elements used as tracers.

[00187] In some embodiments, an "end" modification incorporated into the gNA
is selected from the group consisting of: PEG (polyethyleneglycol), hydrocarbon linkers (including:
heteroatom (0,S,N)-substituted hydrocarbon spacers; halo-substituted hydrocarbon spacers;
keto-, carboxyl-, amido-, thionyl-, carbamoyl-, thionocarbamaoyl-containing hydrocarbon spacers), spermine linkers, dyes including fluorescent dyes (for example fluoresceins, rhodamines, cyanines) attached to linkers such as, for example 6-fluorescein-hexyl, quenchers (for example dabcyl, BHQ) and other labels (for example biotin, digoxigenin, acridine, streptavidin, avidin, peptides and/or proteins). In some embodiments, an "end"
modification comprises a conjugation (or ligation) of the gNA to another molecule comprising an oligonucleotide of deoxynucleotides and/or ribonucleotides, a peptide, a protein, a sugar, an oligosaccharide, a steroid, a lipid, a folic acid, a vitamin and/or other molecule. In certain embodiments, the disclosure provides a chemically-modified gNA in which an "end"
modification (described above) is located internally in the gNA sequence via a linker such as, for example, a 2-(4-butylamidofluorescein)propane-1,3-diol bis(phosphodiester) linker, which is incorporated as a phosphodiester linkage and can be incorporated anywhere between two nucleotides in the gNA.

[00188] In some embodiments, the disclosure provides a chemically-modified gNA
having an end modification comprising a terminal functional group such as an amine, a thiol (or sulfhydryl), a hydroxyl, a carboxyl, carbonyl, thionyl, thiocarbonyl, a carbamoyl, a thiocarbamoyl, a phoshoryl, an alkene, an alkyne, an halogen or a functional group-terminated linker that can be subsequently conjugated to a desired moiety selected from the group consisting of a fluorescent dye, a non-fluorescent label, a tag (for 14C, example biotin, avidin, streptavidin, or moiety containing an isotopic label such as 15N, 13C, deuterium, 3H, 32P 1251 and, the like), an oligonucleotide (comprising deoxynucleotides and/or ribonucleotides, including an aptamer), an amino acid, a peptide, a protein, a sugar, an oligosaccharide, a steroid, a lipid, a folic acid, and a vitamin. The conjugation employs standard chemistry well-known in the art, including but not limited to coupling via N-hydroxysuccinimide, isothiocyanate, DCC (or DCI), and/or any other standard method as described in "Bioconjugate Techniques" by Greg T.
Hermanson, Publisher Eslsevier Science, 3rded. (2013), the contents of which are incorporated herein by reference in its entirety.
i. Complex Formation with CasX Protein

[00189] In some embodiments, a gNA variant has an improved ability to form a complex with a CasX protein (such as a reference CasX or a CasX variant protein) when compared to a reference gRNA. In some embodiments, a gNA variant has an improved affinity for a CasX
protein (such as a reference or variant protein) when compared to a reference gRNA, thereby improving its ability to form a ribonucleoprotein (RNP) complex with the CasX
protein, as described in the Examples. Improving ribonucleoprotein complex formation may, in some embodiments, improve the efficiency with which functional RNPs are assembled.
In some embodiments, greater than 90%, greater than 93%, greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% of RNPs comprising a gNA
variant and a spacer are competent for gene editing of a target nucleic acid.

[00190] Exemplary nucleotide changes that can improve the ability of gNA
variants to form a complex with CasX protein may, in some embodiments, include replacing the scaffold stem with a thermostable stem loop. Without wishing to be bound by any theory, replacing the scaffold stem with a thermostable stem loop could increase the overall binding stability of the gNA
variant with the CasX protein. Alternatively, or in addition, removing a large section of the stem loop could change the gNA variant folding kinetics and make a functional folded gNA easier and quicker to structurally-assemble, for example by lessening the degree to which the gNA
variant can get "tangled" in itself. In some embodiments, choice of scaffold stem loop sequence could change with different spacers that are utilized for the gNA. In some embodiments, scaffold sequence can be tailored to the spacer and therefore the target sequence.
Biochemical assays can be used to evaluate the binding affinity of CasX protein for the gNA variant to form the RNP, including the assays of the Examples. For example, a person of ordinary skill can measure changes in the amount of a fluorescently tagged gNA that is bound to an immobilized CasX
protein, as a response to increasing concentrations of an additional unlabeled "cold competitor"
gNA. Alternatively, or in addition, fluorescence signal can be monitored to or seeing how it changes as different amounts of fluorescently labeled gNA are flowed over immobilized CasX

protein. Alternatively, the ability to form an RNP can be assessed using in vitro cleavage assays against a defined target nucleic acid sequence.
j. gNA Stability

[00191] In some embodiments, a gNA variant has improved stability when compared to a reference gRNA. Increased stability and efficient folding may, in some embodiments, increase the extent to which a gNA variant persists inside a target cell, which may thereby increase the chance of forming a functional RNP capable of carrying out CasX functions such as gene editing. Increased stability of gNA variants may also, in some embodiments, allow for a similar outcome with a lower amount of gNA delivered to a cell, which may in turn reduce the chance of off-target effects during gene editing.

[00192] In other embodiments, the disclosure provides gNA in which the scaffold stem loop and/or the extended stem loop is replaced with a hairpin loop or a thermostable RNA stem loop in which the resulting gNA has increased stability and, depending on the choice of loop, can interact with certain cellular proteins or RNA. In some embodiments, the replacement RNA
loop is selected from MS2, Qf3, Ul hairpin II, Uvsx, PP7, Phage replication loop, Kissing loop a, Kissing loop bl, Kissing loop b2, G quadriplex M3q, G quadriplex telomere basket, Sarcin-ricin loop and Pseudoknots. Sequences of gNA variants including such components are provided in Table 2.

[00193] Guide NA stability can be assessed in a variety of ways, including for example in vitro by assembling the guide, incubating for varying periods of time in a solution that mimics the intracellular environment, and then measuring functional activity via the in vitro cleavage assays described herein. Alternatively, or in addition, gNAs can be harvested from cells at varying time points after initial transfection/transduction of the gNA to determine how long gNA variants persist relative to reference gRNAs.
k. Solubility

[00194] In some embodiments, a gNA variant has improved solubility when compared to a reference gRNA. In some embodiments, a gNA variant has improved solubility of the CasX
protein:gNA RNP when compared to a reference gRNA. In some embodiments, solubility of the CasX protein:gNA RNP is improved by the addition of a ribozyme sequence to a 5' or 3' end of the gNA variant, for example the 5' or 3' of a reference sgRNA. Some ribozymes, such as the M1 ribozyme, can increase solubility of proteins through RNA mediated protein folding.

[00195] Increased solubility of CasX RNPs comprising a gNA variant as described herein can be evaluated through a variety of means known to one of skill in the art, such as by taking densitometry readings on a gel of the soluble fraction of lysed E. coil in which the CasX and gNA variants are expressed.
1. Resistance to Nuclease Activity

[00196] In some embodiments, a gNA variant has improved resistance to nuclease activity compared to a reference gRNA. Without wishing to be bound by any theory, increased resistance to nucleases, such as nucleases found in cells, may for example increase the persistence of a variant gNA in an intracellular environment, thereby improving gene editing.

[00197] Many nucleases are processive, and degrade RNA in a 3' to 5' fashion.
Therefore, in some embodiments the addition of a nuclease resistant secondary structure to one or both termini of the gNA, or nucleotide changes that change the secondary structure of a sgNA, can produce gNA variants with increased resistance to nuclease activity. Resistance to nuclease activity may be evaluated through a variety of methods known to one of skill in the art.
For example, in vitro methods of measuring resistance to nuclease activity may include for example contacting reference gNA and variants with one or more exemplary RNA nucleases and measuring degradation. Alternatively, or in addition, measuring persistence of a gNA
variant in a cellular environment using the methods described herein can indicate the degree to which the gNA
variant is nuclease resistant.
m. Binding Affinity to a Target DNA

[00198] In some embodiments, a gNA variant has improved affinity for the target DNA relative to a reference gRNA. In certain embodiments, a ribonucleoprotein complex comprising a gNA
variant has improved affinity for the target DNA, relative to the affinity of an RNP comprising a reference gRNA. In some embodiments, the improved affinity of the RNP for the target DNA
comprises improved affinity for the target sequence, improved affinity for the PAM sequence, improved ability of the RNP to search DNA for the target sequence, or any combinations thereof In some embodiments, the improved affinity for the target DNA is the result of increased overall DNA binding affinity.

[00199] Without wishing to be bound by theory, it is possible that nucleotide changes in the gNA variant that affect the function of the OBD in the CasX protein may increase the affinity of CasX variant protein binding to the protospacer adjacent motif (PAM), as well as the ability to bind or utilize an increased spectrum of PAM sequences other than the canonical TTC PAM

recognized by the reference CasX protein of SEQ ID NO: 2, including PAM
sequences selected from the group consisting of TTC, ATC, GTC, and CTC, thereby increasing the affinity and diversity of the CasX variant protein for target DNA sequences, thereby increasing the target nucleic acid sequences that can be edited and/or bound, compared to a reference CasX. As described more fully, below, increasing the sequences of the target nucleic acid that can be edited, compared to a reference CasX, refers to both the PAM and the protospacer sequence and their directionality according to the orientation of the non-target strand.
This does not imply that the PAM sequence of the non-target strand, rather than the target strand, is determinative of cleavage or mechanistically involved in target recognition. For example, when reference is to a TTC PAM, it may in fact be the complementary GAA sequence that is required for target cleavage, or it may be some combination of nucleotides from both strands. In the case of the CasX proteins disclosed herein, the PAM is located 5' of the protospacer with at least a single nucleotide separating the PAM from the first nucleotide of the protospacer.
Alternatively, or in addition, changes in the gNA that affect function of the helical I and/or helical II domains that increase the affinity of the CasX variant protein for the target DNA strand can increase the affinity of the CasX RNP comprising the variant gNA for target DNA.
n. Adding or Changing gNA Function

[00200] In some embodiments, gNA variants can comprise larger structural changes that change the topology of the gNA variant with respect to the reference gRNA, thereby allowing for different gNA functionality. For example, in some embodiments a gNA
variant has swapped an endogenous stem loop of the reference gRNA scaffold with a previously identified stable RNA structure or a stem loop that can interact with a protein or RNA binding partner to recruit additional moieties to the CasX or to recruit CasX to a specific location, such as the inside of a viral capsid, that has the binding partner to the said RNA structure. In other scenarios the RNAs may be recruited to each other, as in Kissing loops, such that two CasX
proteins can be co-localized for more effective gene editing at the target DNA sequence. Such RNA
structures may include M52, Q(3, Ul hairpin II, Uvsx, PP7, Phage replication loop, Kissing loop a, Kissing loop bl, Kissing loop b2, G quadriplex M3q, G quadriplex telomere basket, Sarcin-ricin loop, or a Pseudoknot.

[00201] In some embodiments, a gNA variant comprises a terminal fusion partner. The term gNA variant is inclusive of variants that include exogenous sequences such as terminal fusions, or internal insertions. Exemplary terminal fusions may include fusion of the gRNA to a self-cleaving ribozyme or protein binding motif As used herein, a "ribozyme" refers to an RNA or segment thereof with one or more catalytic activities similar to a protein enzyme. Exemplary ribozyme catalytic activities may include, for example, cleavage and/or ligation of RNA, cleavage and/or ligation of DNA, or peptide bond formation. In some embodiments, such fusions could either improve scaffold folding or recruit DNA repair machinery.
For example, a gRNA may in some embodiments be fused to a hepatitis delta virus (HDV) antigenomic ribozyme, HDV genomic ribozyme, hatchet ribozyme (from metagenomic data), env25 pistol ribozyme (representative from Aliistipes putredinis), HH15 Minimal Hammerhead ribozyme, tobacco ringspot virus (TRSV) ribozyme, WT viral Hammerhead ribozyme (and rational variants), or Twisted Sister 1 or RBMX recruiting motif. Hammerhead ribozymes are RNA
motifs that catalyze reversible cleavage and ligation reactions at a specific site within an RNA
molecule. Hammerhead ribozymes include type I, type II and type III hammerhead ribozymes.
The HDV, pistol, and hatchet ribozymes have self-cleaving activities. gNA
variants comprising one or more ribozymes may allow for expanded gNA function as compared to a gRNA
reference. For example, gNAs comprising self-cleaving ribozymes can, in some embodiments, be transcribed and processed into mature gNAs as part of polycistronic transcripts. Such fusions may occur at either the 5' or the 3' end of the gNA. In some embodiments, a gNA variant comprises a fusion at both the 5' and the 3' end, wherein each fusion is independently as described herein. In some embodiments, a gNA variant comprises a phage replication loop or a tetraloop. In some embodiments, a gNA comprises a hairpin loop that is capable of binding a protein. For example, in some embodiments the hairpin loop is an M52, Qf3, Ul hairpin II, Uvsx, or PP7 hairpin loop.

[00202] In some embodiments, a gNA variant comprises one or more RNA aptamers.
As used herein, an "RNA aptamer" refers to an RNA molecule that binds a target with high affinity and high specificity.

[00203] In some embodiments, a gNA variant comprises one or more riboswitches.
As used herein, a "riboswitch" refers to an RNA molecule that changes state upon binding a small molecule.

[00204] In some embodiments, the gNA variant further comprises one or more protein binding motifs. Adding protein binding motifs to a reference gRNA or gNA variant of the disclosure may, in some embodiments, allow a CasX RNP to associate with additional proteins, which can for example add the functionality of those proteins to the CasX RNP.

IV. CasX Proteins for Modifying a Target Nucleic Acid

[00205] The term "CasX protein", as used herein, refers to a family of proteins, and encompasses all naturally occurring CasX proteins, proteins that share at least 50% identity to naturally occurring CasX proteins, as well as CasX variants possessing one or more improved characteristics relative to a naturally-occurring reference CasX protein.
Exemplary improved characteristics of the CasX variant embodiments include, but are not limited to improved folding of the variant, improved binding affinity to the gNA, improved binding affinity to the target nucleic acid, improved ability to utilize a greater spectrum of PAM sequences in the editing and/or binding of target DNA, improved unwinding of the target DNA, increased editing activity, improved editing efficiency, improved editing specificity, increased percentage of a eukaryotic genome that can be efficiently edited, increased activity of the nuclease, increased target strand loading for double strand cleavage, decreased target strand loading for single strand nicking, decreased off-target cleavage, improved binding of the non-target strand of DNA, improved protein stability, improved protein:gNA (RNP) complex stability, improved protein solubility, improved protein:gNA (RNP) complex solubility, improved protein yield, improved protein expression, and improved fusion characteristics, as described more fully, below. In the foregoing embodiments, the one or more of the improved characteristics of the CasX variant is at least about 1.1 to about 100,000-fold improved relative to the reference CasX
protein of SEQ ID
NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 when assayed in a comparable fashion. In other embodiments, the improvement is at least about 1.1-fold, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, at least about 1000-fold, at least about 5000-fold, at least about 10,000-fold, or at least about 100,000-fold compared to the reference CasX protein of SEQ ID NO: 1, SEQ ID
NO: 2, or SEQ
ID NO: 3 when assayed in a comparable fashion.

[00206] The term CasX variant is inclusive of variants that are fusion proteins, i.e. the CasX is "fused to" a heterologous sequence. This includes CasX variants comprising CasX variant sequences and N-terminal, C-terminal, or internal fusions of the CasX to a heterologous protein or domain thereof.

[00207] CasX proteins of the disclosure comprise at least one of the following domains: a non-target strand binding (NTSB) domain, a target strand loading (TSL) domain, a helical I domain, a helical II domain, an oligonucleotide binding domain (OBD), and a RuvC DNA
cleavage domain (the last of which may be modified or deleted in a catalytically dead CasX variant), described more fully, below. Additionally, the CasX variant proteins of the disclosure have an enhanced ability to efficiently edit and/or bind target DNA utilizing PAM
sequences selected from TTC, ATC, GTC, or CTC, compared to wild-type reference CasX proteins. In the foregoing, the PAM sequence is located at least 1 nucleotide 5' to the non-target strand of the protospacer having identity with the targeting sequence of the gNA in a assay system compared to the editing efficiency and/or binding of an RNP comprising a reference CasX
protein in a comparable assay system.

[00208] In some cases, the CasX protein is a naturally-occurring protein (e.g., naturally occurs in and is isolated from prokaryotic cells). In other embodiments, the CasX
protein is not a naturally-occurring protein (e.g., the CasX protein is a CasX variant protein, a chimeric protein, and the like). A naturally-occurring CasX protein (referred to herein as a "reference CasX
protein") functions as an endonuclease that catalyzes a double strand break at a specific sequence in a targeted double-stranded DNA (dsDNA). The sequence specificity is provided by the targeting sequence of the associated gNA to which it is complexed, which hybridizes to a target sequence within the target nucleic acid.

[00209] In some embodiments, a CasX protein can bind and/or modify (e.g., cleave, nick, methylate, demethylate, etc.) a target nucleic acid and/or a polypeptide associated with target nucleic acid (e.g., methylation or acetylation of a histone tail). In some embodiments, the CasX
protein is catalytically dead (dCasX) but retains the ability to bind a target nucleic acid. An exemplary catalytically dead CasX protein comprises one or more mutations in the active site of the RuvC domain of the CasX protein. In some embodiments, a catalytically dead CasX protein comprises substitutions at residues 672, 769 and/or 935 of SEQ ID NO: 1. In one embodiment, a catalytically dead CasX protein comprises substitutions of D672A, E769A and/or D935A in a reference CasX protein of SEQ ID NO: 1. In other embodiments, a catalytically dead CasX
protein comprises substitutions at amino acids 659, 756 and/or 922 in a reference CasX protein of SEQ ID NO: 2. In some embodiments, a catalytically dead CasX protein comprises D659A, E756A and/or D922A substitutions in a reference CasX protein of SEQ ID NO: 2.
In further embodiments, a catalytically dead CasX protein comprises deletions of all or part of the RuvC
domain of the CasX protein. It will be understood that the same foregoing substitutions can similarly be introduced into the CasX variants of the disclosure, resulting in a dCasX variant. In one embodiment, all or a portion of the RuvC domain is deleted from the CasX
variant, resulting in a dCasX variant. Catalytically inactive dCasX variant proteins can, in some embodiments, be used for base editing or epigenetic modifications. With a higher affinity for DNA, in some embodiments, catalytically inactive dCasX variant proteins can, relative to catalytically active CasX, find their target nucleic acid faster, remain bound to target nucleic acid for longer periods of time, bind target nucleic acid in a more stable fashion, or a combination thereof, thereby improving the function of the catalytically dead CasX variant protein.
a. Non-Target Strand Binding Domain

[00210] The reference CasX proteins of the disclosure comprise a non-target strand binding domain (NTSBD). The NTSBD is a domain not previously found in any Cas proteins; for example this domain is not present in Cas proteins such as Cas9, Cas12a/Cpfl, Cas13, Cas14, CASCADE, CSM, or CSY. Without being bound to theory or mechanism, a NTSBD in a CasX
allows for binding to the non-target DNA strand and may aid in unwinding of the non-target and target strands. The NTSBD is presumed to be responsible for the unwinding, or the capture, of a non-target DNA strand in the unwound state. The NTSBD is in direct contact with the non-target strand in CryoEM model structures derived to date and may contain a non-canonical zinc finger domain. The NTSBD may also play a role in stabilizing DNA during unwinding, guide RNA
invasion and R-loop formation. In some embodiments, an exemplary NTSBD
comprises amino acids 101-191 of SEQ ID NO: 1 or amino acids 103-192 of SEQ ID NO: 2. In some embodiments, the NTSBD of a reference CasX protein comprises a four-stranded beta sheet.
b. Target Strand Loading Domain

[00211] The reference CasX proteins of the disclosure comprise a Target Strand Loading (TSL) domain. The TSL domain is a domain not found in certain Cas proteins such as Cas9, CASCADE, CSM, or CSY. Without wishing to be bound by theory or mechanism, it is thought that the TSL domain is responsible for aiding the loading of the target DNA
strand into the RuvC active site of a CasX protein. In some embodiments, the TSL acts to place or capture the target-strand in a folded state that places the scissile phosphate of the target strand DNA
backbone in the RuvC active site. The TSL comprises a cys4 (C)OX (SEQ ID NO:
246, CXXC
(SEQ ID NO: 246) zinc finger/ribbon domain that is separated by the bulk of the TSL. In some embodiments, an exemplary TSL comprises amino acids 825-934 of SEQ ID NO: 1 or amino acids 813-921 of SEQ ID NO: 2.
c. Helical I Domain

[00212] The reference CasX proteins of the disclosure comprise a helical I
domain. Certain Cas proteins other than CasX have domains that may be named in a similar way.
However, in some embodiments, the helical I domain of a CasX protein comprises one or more unique structural features, or comprises a unique sequence, or a combination thereof, compared to non-CasX proteins. For example, in some embodiments, the helical I domain of a CasX protein comprises one or more unique secondary structures compared to domains in other Cas proteins that may have a similar name. For example, in some embodiments the helical I
domain in a CasX protein comprises one or more alpha helices of unique structure and sequence in arrangement, number and length compared to other CRISPR proteins. In certain embodiments, the helical I domain is responsible for interacting with the bound DNA and spacer of the guide RNA. Without wishing to be bound by theory, it is thought that in some cases the helical I
domain may contribute to binding of the protospacer adjacent motif (PAM). In some embodiments, an exemplary helical I domain comprises amino acids 57-100 and 192-332 of SEQ ID NO: 1, or amino acids 59-102 and 193-333 of SEQ ID NO: 2. In some embodiments, the helical I domain of a reference CasX protein comprises one or more alpha helices.
d. Helical II Domain

[00213] The reference CasX proteins of the disclosure comprise a helical II
domain. Certain Cas proteins other than CasX have domains that may be named in a similar way.
However, in some embodiments, the helical II domain of a CasX protein comprises one or more unique structural features, or a unique sequence, or a combination thereof, compared to domains in other Cas proteins that may have a similar name. For example, in some embodiments, the helical II domain comprises one or more unique structural alpha helical bundles that align along the target DNA:guide RNA channel. In some embodiments, in a CasX comprising a helical II
domain, the target strand and guide RNA interact with helical II (and the helical I domain, in some embodiments) to allow RuvC domain access to the target DNA. The helical II domain is responsible for binding to the guide RNA scaffold stem loop as well as the bound DNA. In some embodiments, an exemplary helical II domain comprises amino acids 333-509 of SEQ ID NO: 1, or amino acids 334-501 of SEQ ID NO: 2.
e. Oligonucleotide Binding Domain

[00214] The reference CasX proteins of the disclosure comprise an Oligonucleotide Binding Domain (OBD). Certain Cas proteins other than CasX have domains that may be named in a similar way. However, in some embodiments, the OBD comprises one or more unique functional features, or comprises a sequence unique to a CasX protein, or a combination thereof.
For example, in some embodiments the bridged helix (BH), helical I domain, helical II domain, and Oligonucleotide Binding Domain (OBD) together are responsible for binding of a CasX
protein to the guide RNA. Thus, for example, in some embodiments the OBD is unique to a CasX protein in that it interacts functionally with a helical I domain, or a helical II domain, or both, each of which may be unique to a CasX protein as described herein.
Specifically, in CasX
the OBD largely binds the RNA triplex of the guide RNA scaffold. The OBD may also be responsible for binding to the protospacer adjacent motif (PAM). An exemplary OBD domain comprises amino acids 1-56 and 510-660 of SEQ ID NO: 1, or amino acids 1-58 and 502-647 of SEQ ID NO: 2.
f. RuvC DNA Cleavage Domain

[00215] The reference CasX proteins of the disclosure comprise a RuvC domain, that includes 2 partial RuvC domains (RuvC-I and RuvC-II). The RuvC domain is the ancestral domain of all type 12 CRISPR proteins. The RuvC domain originates from a TNPB (transposase B) like transposase. Similar to other RuvC domains, the CasX RuvC domain has a DED
catalytic triad that is responsible for coordinating a magnesium (Mg) ion and cleaving DNA. In some embodiments, the RuvC has a DED motif active site that is responsible for cleaving both strands of DNA (one by one, most likely the non-target strand first at 11-14 nucleotides (nt) into the targeted sequence and then the target strand next at 2-4 nucleotides after the target sequence).
Specifically in CasX, the RuvC domain is unique in that it is also responsible for binding the guide RNA scaffold stem loop that is critical for CasX function. An exemplary RuvC domain comprises amino acids 661-824 and 935-986 of SEQ ID NO: 1, or amino acids 648-812 and 922-978 of SEQ ID NO: 2.
g. Reference CasX Proteins

[00216] The disclosure provides reference CasX proteins. In some embodiments, a reference CasX protein is a naturally-occurring protein. For example, reference CasX
proteins can be isolated from naturally occurring prokaryotes, such as Deltaproteobacteria, Planctomycetes, or Candidatus Sungbacteria species. A reference CasX protein (sometimes referred to herein as a reference CasX polypeptide) is a type II CRISPR/Cas endonuclease belonging to the CasX
(sometimes referred to as Cas12e) family of proteins that is capable of interacting with a guide NA to form a ribonucleoprotein (RNP) complex. In some embodiments, the RNP
complex comprising the reference CasX protein can be targeted to a particular site in a target nucleic acid via base pairing between the targeting sequence (or spacer) of the gNA and a target sequence in the target nucleic acid. In some embodiments, the RNP comprising the reference CasX protein is capable of cleaving target DNA. In some embodiments, the RNP comprising the reference CasX protein is capable of nicking target DNA. In some embodiments, the RNP
comprising the reference CasX protein is capable of editing target DNA, for example in those embodiments where the reference CasX protein is capable of cleaving or nicking DNA, followed by non-homologous end joining (NHEJ), homology-directed repair (HDR), homology-independent targeted integration (HITT), micro-homology mediated end joining (MMEJ), single strand annealing (SSA) or base excision repair (BER). In some embodiments, the RNP
comprising the CasX protein is a catalytically dead (is catalytically inactive or has substantially no cleavage activity) CasX protein (dCasX), but retains the ability to bind the target DNA, described more fully, supra.

[00217] In some cases, a reference CasX protein is isolated or derived from Deltaproteobacteria. In some embodiments, a CasX protein comprises a sequence at least 50%
identical, at least 60% identical, at least 65% identical, at least 70%
identical, at least 75%
identical, at least 80% identical, at least 81% identical, at least 82%
identical, at least 83%
identical, at least 84% identical, at least 85% identical, at least 86%
identical, at least 86%
identical, at least 87% identical, at least 88% identical, at least 89%
identical, at least 89%
identical, at least 90% identical, at least 91% identical, at least 92%
identical, at least 93%
identical, at least 94% identical, at least 95% identical, at least 96%
identical, at least 97%
identical, at least 98% identical, at least 99% identical, at least 99.5%
identical or 100% identical to a sequence of:

961 SGKQPFVGAW QAFYKRRLKE VWKPNA (SEQ ID NO: 1).

[00218] In some cases, a reference CasX protein is isolated or derived from Planctomycetes. In some embodiments, a CasX protein comprises a sequence at least 50% identical, at least 60%

identical, at least 65% identical, at least 70% identical, at least 75%
identical, at least 80%
identical, at least 81% identical, at least 82% identical, at least 83%
identical, at least 84%
identical, at least 85% identical, at least 86% identical, at least 86%
identical, at least 87%
identical, at least 88% identical, at least 89% identical, at least 89%
identical, at least 90%
identical, at least 91% identical, at least 92% identical, at least 93%
identical, at least 94%
identical, at least 95% identical, at least 96% identical, at least 97%
identical, at least 98%
identical, at least 99% identical, at least 99.5% identical or 100% identical to a sequence of:

961 TWQSFYRKKL KEVWKPAV (SEQ ID NO: 2).

[00219] In some embodiments, the CasX protein comprises the sequence of SEQ ID
NO: 2, or at least 60% similarity thereto. In some embodiments, the CasX protein comprises the sequence of SEQ ID NO: 2, or at least 80% similarity thereto. In some embodiments, the CasX protein comprises the sequence of SEQ ID NO: 2, or at least 90% similarity thereto. In some embodiments, the CasX protein comprises the sequence of SEQ ID NO: 2, or at least 95%
similarity thereto. In some embodiments, the CasX protein consists of the sequence of SEQ ID
NO: 2. In some embodiments, the CasX protein comprises or consists of a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40 or at least 50 mutations relative to the sequence of SEQ ID
NO: 2. These mutations can be insertions, deletions, amino acid substitutions, or any combinations thereof.

[00220] In some cases, a reference CasX protein is isolated or derived from Candidatus Sungbacteria. In some embodiments, a CasX protein comprises a sequence at least 50%
identical, at least 60% identical, at least 65% identical, at least 70%
identical, at least 75%
identical, at least 80% identical, at least 81% identical, at least 82%
identical, at least 83%

identical, at least 84% identical, at least 85% identical, at least 86%
identical, at least 86%
identical, at least 87% identical, at least 88% identical, at least 89%
identical, at least 89%
identical, at least 90% identical, at least 91% identical, at least 92%
identical, at least 93%
identical, at least 94% identical, at least 95% identical, at least 96%
identical, at least 97%
identical, at least 98% identical, at least 99% identical, at least 99.5%
identical or 100% identical to a sequence of 841 SLIRRLPDTD TPPTP (SEQ ID NO: 3).

[00221] In some embodiments, the CasX protein comprises the sequence of SEQ ID
NO: 3, or at least 60% similarity thereto. In some embodiments, the CasX protein comprises the sequence of SEQ ID NO: 3, or at least 80% similarity thereto. In some embodiments, the CasX protein comprises the sequence of SEQ ID NO: 3, or at least 90% similarity thereto. In some embodiments, the CasX protein comprises the sequence of SEQ ID NO: 3, or at least 95%
similarity thereto. In some embodiments, the CasX protein consists of the sequence of SEQ ID
NO: 3. In some embodiments, the CasX protein comprises or consists of a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40 or at least 50 mutations relative to the sequence of SEQ ID
NO: 3. These mutations can be insertions, deletions, amino acid substitutions, or any combinations thereof h. CasX Variant Proteins

[00222] The present disclosure provides variants of a reference CasX protein (interchangeably referred to herein as "CasX variant" or "CasX variant protein"), wherein the CasX variants comprise at least one modification in at least one domain relative to the reference CasX protein, including but not limited to the sequences of SEQ ID NOS:1-3. In some embodiments, the CasX variant exhibits at least one improved characteristic compared to the reference CasX

protein. All variants that improve one or more functions or characteristics of the CasX variant protein when compared to a reference CasX protein described herein are envisaged as being within the scope of the disclosure. In some embodiments, the modification is a mutation in one or more amino acids of the reference CasX. In other embodiments, the modification is a substitution of one or more domains of the reference CasX with one or more domains from a different CasX. In some embodiments, insertion includes the insertion of a part or all of a domain from a different CasX protein. Mutations can occur in any one or more domains of the reference CasX protein, and may include, for example, deletion of part or all of one or more domains, or one or more amino acid substitutions, deletions, or insertions in any domain of the reference CasX protein. The domains of CasX proteins include the non-target strand binding (NTSB) domain, the target strand loading (TSL) domain, the helical I domain, the helical II
domain, the oligonucleotide binding domain (OBD), and the RuvC DNA cleavage domain. Any change in amino acid sequence of a reference CasX protein that leads to an improved characteristic of the CasX protein is considered a CasX variant protein of the disclosure. For example, CasX variants can comprise one or more amino acid substitutions, insertions, deletions, or swapped domains, or any combinations thereof, relative to a reference CasX protein sequence.

[00223] In some embodiments, the CasX variant protein comprises at least one modification in at least each of two domains of the reference CasX protein, including the sequences of SEQ ID
NOS: 1-3. In some embodiments, the CasX variant protein comprises at least one modification in at least 2 domains, in at least 3 domains, at least 4 domains or at least 5 domains of the reference CasX protein. In some embodiments, the CasX variant protein comprises two or more modifications in at least one domain of the reference CasX protein. In some embodiments, the CasX variant protein comprises at least two modifications in at least one domain of the reference CasX protein, at least three modifications in at least one domain of the reference CasX protein or at least four modifications in at least one domain of the reference CasX
protein. In some embodiments, wherein the CasX variant comprises two or more modifications compared to a reference CasX protein, each modification is made in a domain independently selected from the group consisting of a NTSBD, TSLD, helical I domain, helical II domain, OBD, and RuvC DNA
cleavage domain.

[00224] In some embodiments, the at least one modification of the CasX variant protein comprises a deletion of at least a portion of one domain of the reference CasX
protein. In some embodiments, the deletion is in the NTSBD, TSLD, helical I domain, helical II
domain, OBD, or RuvC DNA cleavage domain.

[00225] Suitable mutagenesis methods for generating CasX variant proteins of the disclosure may include, for example, Deep Mutational Evolution (DME), deep mutational scanning (DMS), error prone PCR, cassette mutagenesis, random mutagenesis, staggered extension PCR, gene shuffling, or domain swapping. Exemplary methods for the generation of CasX
variants with improved characteristics are provided in the Examples, below. In some embodiments, the CasX
variants are designed, for example by selecting one or more desired mutations in a reference CasX. In certain embodiments, the activity of a reference CasX protein is used as a benchmark against which the activity of one or more CasX variants are compared, thereby measuring improvements in function of the CasX variants. Exemplary improvements of CasX
variants include, but are not limited to, improved folding of the variant, improved binding affinity to the gNA, improved binding affinity to the target DNA, improved ability to utilize a greater spectrum of PAM sequences in the editing or binding of target DNA, improved unwinding of the target DNA, increased editing activity, improved editing efficiency, improved editing specificity, increased activity of the nuclease, increased target strand loading for double strand cleavage, decreased target strand loading for single strand nicking, decreased off-target cleavage, improved binding of the non-target strand of DNA, improved protein stability, improved CasX:gNA (RNP) complex stability, improved protein solubility, improved CasX:gNA (RNP) complex solubility, improved protein yield, improved protein expression, and improved fusion characteristics, as described more fully, below.

[00226] In some embodiments of the CasX variants described herein, the at least one modification comprises: (a) a substitution of 1 to 100 consecutive or non-consecutive amino acids in the CasX variant; (b) a deletion of 1 to 100 consecutive or non-consecutive amino acids in the CasX variant; (c) an insertion of 1 to 100 consecutive or non-consecutive amino acids in the CasX; or (d) any combination of (a)-(c). In some embodiments, the at least one modification comprises: (a) a substitution of 5-10 consecutive or non-consecutive amino acids in the CasX
variant; (b) a deletion of 1-5 consecutive or non-consecutive amino acids in the CasX variant; (c) an insertion of 1-5 consecutive or non-consecutive amino acids in the CasX; or (d) any combination of (a)-(c).

[00227] In some embodiments, the CasX variant protein comprises or consists of a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40 or at least 50 mutations relative to the sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. These mutations can be insertions, deletions, amino acid substitutions, or any combinations thereof.

[00228] In some embodiments, the CasX variant protein comprises at least one amino acid substitution in at least one domain of a reference CasX protein. In some embodiments, the CasX
variant protein comprises at least about 1-4 amino acid substitutions, 1-10 amino acid substitutions, 1-20 amino acid substitutions, 1-30 amino acid substitutions, 1-40 amino acid substitutions, 1-50 amino acid substitutions, 1-60 amino acid substitutions, 1-70 amino acid substitutions, 1-80 amino acid substitutions, 1-90 amino acid substitutions, 1-100 amino acid substitutions, 2-10 amino acid substitutions, 2-20 amino acid substitutions, 2-30 amino acid substitutions, 3-10 amino acid substitutions, 3-20 amino acid substitutions, 3-30 amino acid substitutions, 4-10 amino acid substitutions, 4-20 amino acid substitutions, 3-300 amino acid substitutions, 5-10 amino acid substitutions, 5-20 amino acid substitutions, 5-30 amino acid substitutions, 10-50 amino acid substitutions, or 20-50 amino acid substitutions, relative to a reference CasX protein. In some embodiments, the CasX variant protein comprises at least about 100 amino acid substitutions relative to a reference CasX protein. In some embodiments, the CasX variant protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions relative to a reference CasX protein. In some embodiments, the CasX variant protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions in a single domain relative to the reference CasX
protein. In some embodiments, the amino acid substitutions are conservative substitutions. In other embodiments, the substitutions are non-conservative; e.g., a polar amino acid is substituted for a non-polar amino acid, or vice versa.

[00229] In some embodiments, a CasX variant protein comprises 1 amino acid substitution, 2-3 consecutive amino acid substitutions, 2-4 consecutive amino acid substitutions, 2-5 consecutive amino acid substitutions, 2-6 consecutive amino acid substitutions, 2-7 consecutive amino acid substitutions, 2-8 consecutive amino acid substitutions, 2-9 consecutive amino acid substitutions, 2-10 consecutive amino acid substitutions, 2-20 consecutive amino acid substitutions, 2-30 consecutive amino acid substitutions, 2-40 consecutive amino acid substitutions, 2-50 consecutive amino acid substitutions, 2-60 consecutive amino acid substitutions, 2-70 consecutive amino acid substitutions, 2-80 consecutive amino acid substitutions, 2-90 consecutive amino acid substitutions, 2-100 consecutive amino acid substitutions, 3-10 consecutive amino acid substitutions, 3-20 consecutive amino acid substitutions, 3-30 consecutive amino acid substitutions, 4-10 consecutive amino acid substitutions, 4-20 consecutive amino acid substitutions, 3-300 consecutive amino acid substitutions, 5-10 consecutive amino acid substitutions, 5-20 consecutive amino acid substitutions, 5-30 consecutive amino acid substitutions, 10-50 consecutive amino acid substitutions or 20-50 consecutive amino acid substitutions relative to a reference CasX protein. In some embodiments, a CasX variant protein comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 consecutive amino acid substitutions. In some embodiments, a CasX variant protein comprises a substitution of at least about 100 consecutive amino acids. As used herein "consecutive amino acids" refer to amino acids that are contiguous in the primary sequence of a polypeptide.

[00230] In some embodiments, a CasX variant protein comprises two or more substitutions relative to a reference CasX protein, and the two or more substitutions are not in consecutive amino acids of the reference CasX sequence. For example, a first substitution may be in a first domain of the reference CasX protein, and a second substitution may be in a second domain of the reference CasX protein. In some embodiments, a CasX variant protein comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 non-consecutive substitutions relative to a reference CasX protein. In some embodiments, a CasX variant protein comprises at least 20 non-consecutive substitutions relative to a reference CasX protein. Each non-consecutive substitution may be of any length of amino acids described herein, e.g., 1-4 amino acids, 1-10 amino acids, and the like. In some embodiments, the two or more substitutions relative to the reference CasX
protein are not the same length, for example, one substitution is one amino acid and a second substitution is three amino acids. In some embodiments, the two or more substitutions relative to the reference CasX protein are the same length, for example both substitutions are two consecutive amino acids in length.

[00231] Any amino acid can be substituted for any other amino acid in the substitutions described herein. The substitution can be a conservative substitution (e.g., a basic amino acid is substituted for another basic amino acid). The substitution can be a non-conservative substitution (e.g., a basic amino acid is substituted for an acidic amino acid or vice versa). For example, a proline in a reference CasX protein can be substituted for any of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine or valine to generate a CasX variant protein of the disclosure.

[00232] In some embodiments, a CasX variant protein comprises at least one amino acid deletion relative to a reference CasX protein. In some embodiments, a CasX
variant protein comprises a deletion of 1-4 amino acids, 1-10 amino acids, 1-20 amino acids, 1-30 amino acids, 1-40 amino acids, 1-50 amino acids, 1-60 amino acids, 1-70 amino acids, 1-80 amino acids, 1-90 amino acids, 1-100 amino acids, 2-10 amino acids, 2-20 amino acids, 2-30 amino acids, 3-10 amino acids, 3-20 amino acids, 3-30 amino acids, 4-10 amino acids, 4-20 amino acids, 3-300 amino acids, 5-10 amino acids, 5-20 amino acids, 5-30 amino acids, 10-50 amino acids or 20-50 amino acids relative to a reference CasX protein. In some embodiments, a CasX
variant comprises a deletion of at least about 100 consecutive amino acids relative to a reference CasX
protein. In some embodiments, a CasX variant protein comprises a deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 or 100 consecutive amino acids relative to a reference CasX
protein. In some embodiments, a CasX variant protein comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids.

[00233] In some embodiments, a CasX variant protein comprises two or more deletions relative to a reference CasX protein, and the two or more deletions are not consecutive amino acids. For example, a first deletion may be in a first domain of the reference CasX
protein, and a second deletion may be in a second domain of the reference CasX protein. In some embodiments, a CasX variant protein comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 non-consecutive deletions relative to a reference CasX protein. In some embodiments, a CasX
variant protein comprises at least 20 non-consecutive deletions relative to a reference CasX
protein. Each non-consecutive deletion may be of any length of amino acids described herein, e.g., 1-4 amino acids, 1-10 amino acids, and the like.

[00234] In some embodiments, the CasX variant protein comprises at least one amino acid insertion. In some embodiments, a CasX variant protein comprises an insertion of 1 amino acid, an insertion of 2-3 consecutive amino acids, 2-4 consecutive amino acids, 2-5 consecutive amino acids, 2-6 consecutive amino acids, 2-7 consecutive amino acids, 2-8 consecutive amino acids, 2-9 consecutive amino acids, 2-10 consecutive amino acids, 2-20 consecutive amino acids, 2-30 consecutive amino acids, 2-40 consecutive amino acids, 2-50 consecutive amino acids, 2-60 consecutive amino acids, 2-70 consecutive amino acids, 2-80 consecutive amino acids, 2-90 consecutive amino acids, 2-100 consecutive amino acids, 3-10 consecutive amino acids, 3-20 consecutive amino acids, 3-30 consecutive amino acids, 4-10 consecutive amino acids, 4-20 consecutive amino acids, 3-300 consecutive amino acids, 5-10 consecutive amino acids, 5-20 consecutive amino acids, 5-30 consecutive amino acids, 10-50 consecutive amino acids or 20-50 consecutive amino acids relative to a reference CasX protein. In some embodiments, the CasX
variant protein comprises an insertion of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 consecutive amino acids. In some embodiments, a CasX variant protein comprises an insertion of at least about 100 consecutive amino acids.

[00235] In some embodiments, a CasX variant protein comprises two or more insertions relative to a reference CasX protein, and the two or more insertions are not consecutive amino acids of the sequence. For example, a first insertion may be in a first domain of the reference CasX protein, and a second insertion may be in a second domain of the reference CasX protein.
In some embodiments, a CasX variant protein comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 non-consecutive insertions relative to a reference CasX protein. In some embodiments, a CasX variant protein comprises at least 10 to about 20 or more non-consecutive insertions relative to a reference CasX protein. Each non-consecutive insertion may be of any length of amino acids described herein, e.g., 1-4 amino acids, 1-10 amino acids, and the like.

[00236] Any amino acid, or combination of amino acids, can be inserted as described herein.
For example, a proline, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine or valine or any combination thereof can be inserted into a reference CasX
protein of the disclosure to generate a CasX variant protein.

[00237] Any permutation of the substitution, insertion and deletion embodiments described herein can be combined to generate a CasX variant protein of the disclosure.
For example, a CasX variant protein can comprise at least one substitution and at least one deletion relative to a reference CasX protein sequence, at least one substitution and at least one insertion relative to a reference CasX protein sequence, at least one insertion and at least one deletion relative to a reference CasX protein sequence, or at least one substitution, one insertion and one deletion relative to a reference CasX protein sequence.

[00238] In some embodiments, the CasX variant protein has at least about 60%
sequence similarity, at least 70% similarity, at least 80% similarity, at least 85%
similarity, at least 86%
similarity, at least 87% similarity, at least 88% similarity, at least 89%
similarity, at least 90%
similarity, at least 91% similarity, at least 92% similarity, at least 93%
similarity, at least 94%
similarity, at least 95% similarity, at least 96% similarity, at least 97%
similarity, at least 98%
similarity, at least 99% similarity, at least 99.5% similarity, at least 99.6%
similarity, at least 99.7% similarity, at least 99.8% similarity or at least 99.9% similarity to one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[00239] In some embodiments, the CasX variant protein has at least about 60%
sequence similarity to SEQ ID NO: 2 or a portion thereof. In some embodiments, the CasX
variant protein comprises a substitution of Y789T of SEQ ID NO: 2, a deletion of P793 of SEQ
ID NO: 2, a substitution of Y789D of SEQ ID NO: 2, a substitution of T725 of SEQ ID NO: 2, a substitution of I546V of SEQ ID NO: 2, a substitution of E552A of SEQ ID NO: 2, a substitution of A636D
of SEQ ID NO: 2, a substitution of F5365 of SEQ ID NO:2, a substitution of A708K of SEQ ID
NO: 2, a substitution of Y797L of SEQ ID NO: 2, a substitution of L792G SEQ ID
NO: 2, a substitution of A739V of SEQ ID NO: 2, a substitution of G791M of SEQ ID NO:
2, an insertion of A at position 661of SEQ ID NO: 2, a substitution of A788W of SEQ
ID NO: 2, a substitution of K390R of SEQ ID NO: 2, a substitution of A751S of SEQ ID NO:
2, a substitution of E385A of SEQ ID NO: 2, an insertion of P at position 696 of SEQ ID NO: 2, an insertion of M at position 773 of SEQ ID NO: 2, a substitution of G695H of SEQ
ID NO: 2, an insertion of AS at position 793 of SEQ ID NO: 2, an insertion of AS at position 795 of SEQ ID
NO: 2, a substitution of C477R of SEQ ID NO: 2, a substitution of C477K of SEQ
ID NO: 2, a substitution of C479A of SEQ ID NO: 2, a substitution of C479L of SEQ ID NO:
2, a substitution of I55F of SEQ ID NO: 2, a substitution of K21OR of SEQ ID NO: 2, a substitution of C233S of SEQ ID NO: 2, a substitution of D23 1N of SEQ ID NO: 2, a substitution of Q33 8E
of SEQ ID NO: 2, a substitution of Q338R of SEQ ID NO: 2, a substitution of L379R of SEQ ID
NO: 2, a substitution of K390R of SEQ ID NO: 2, a substitution of L481Q of SEQ
ID NO: 2, a substitution of F495S of SEQ ID NO:2, a substitution of D600N of SEQ ID NO: 2, a substitution of T886K of SEQ ID NO: 2, a substitution of A739V of SEQ ID NO: 2, a substitution of K460N
of SEQ ID NO: 2, a substitution of I199F of SEQ ID NO: 2, a substitution of G492P of SEQ ID
NO: 2, a substitution of T1531 of SEQ ID NO: 2, a substitution of R591I of SEQ
ID NO: 2, an insertion of AS at position 795 of SEQ ID NO: 2, an insertion of AS at position 796 of SEQ ID
NO:2, an insertion of L at position 889 of SEQ ID NO: 2, a substitution of E121D of SEQ ID
NO: 2, a substitution of S270W of SEQ ID NO: 2, a substitution of E712Q of SEQ
ID NO: 2, a substitution of K942Q of SEQ ID NO: 2, a substitution of E552K of SEQ ID NO:2, a substitution of K25Q of SEQ ID NO: 2, a substitution of N47D of SEQ ID NO: 2, an insertion of T at position 696 of SEQ ID NO: 2, a substitution of L685I of SEQ ID NO: 2, a substitution of N880D of SEQ ID NO: 2, a substitution of Q102R of SEQ ID NO: 2, a substitution of M734K

of SEQ ID NO: 2, a substitution of A7245 of SEQ ID NO: 2, a substitution of T704K of SEQ ID
NO: 2, a substitution of P224K of SEQ ID NO: 2, a substitution of K25R of SEQ
ID NO: 2, a substitution of M29E of SEQ ID NO: 2, a substitution of H152D of SEQ ID NO: 2, a substitution of 5219R of SEQ ID NO: 2, a substitution of E475K of SEQ ID NO:
2, a substitution of G226R of SEQ ID NO: 2, a substitution of A377K of SEQ ID NO:
2, a substitution of E480K of SEQ ID NO: 2, a substitution of K416E of SEQ ID NO:
2, a substitution of H164R of SEQ ID NO: 2, a substitution of K767R of SEQ ID NO:
2, a substitution of I7F of SEQ ID NO: 2, a substitution of M29R of SEQ ID NO: 2, a substitution of H435R of SEQ ID NO: 2, a substitution of E385Q of SEQ ID NO: 2, a substitution of E385K of SEQ ID NO: 2, a substitution of I279F of SEQ ID NO: 2, a substitution of D4895 of SEQ ID
NO: 2, a substitution of D732N of SEQ ID NO: 2, a substitution of A739T of SEQ
ID NO: 2, a substitution of W885R of SEQ ID NO: 2, a substitution of E53K of SEQ ID NO: 2, a substitution of A238T of SEQ ID NO: 2, a substitution of P283Q of SEQ ID NO:
2, a substitution of E292K of SEQ ID NO: 2, a substitution of Q628E of SEQ ID NO:
2, a substitution of R388Q of SEQ ID NO: 2, a substitution of G791M of SEQ ID NO:
2, a substitution of L792K of SEQ ID NO: 2, a substitution of L792E of SEQ ID NO:
2, a substitution of M779N of SEQ ID NO: 2, a substitution of G27D of SEQ ID NO: 2, a substitution of K955R of SEQ ID NO: 2, a substitution of 5867R of SEQ ID NO:
2, a substitution of R693I of SEQ ID NO: 2, a substitution of F189Y of SEQ ID NO:
2, a substitution of V635M of SEQ ID NO: 2, a substitution of F399L of SEQ ID NO: 2, a substitution of E498K
of SEQ ID NO: 2, a substitution of E386R of SEQ ID NO: 2, a substitution of V254G of SEQ
ID NO: 2, a substitution of P793S of SEQ ID NO: 2, a substitution of K188E of SEQ ID NO: 2, a substitution of QT945KI of SEQ ID NO: 2, a substitution of T620P of SEQ ID
NO: 2, a substitution of T946P of SEQ ID NO: 2, a substitution of TT949PP of SEQ ID NO:
2, a substitution of N952T of SEQ ID NO: 2, a substitution of K682E of SEQ ID NO:
2, a substitution of K975R of SEQ ID NO: 2, a substitution of L212P of SEQ ID NO:
2, a substitution of E292R of SEQ ID NO: 2, a substitution of 1303K of SEQ ID NO:
2, a substitution of C349E of SEQ ID NO: 2, a substitution of E385P of SEQ ID NO:
2, a substitution of E386N of SEQ ID NO: 2, a substitution of D387K of SEQ ID NO:
2, a substitution of L404K of SEQ ID NO: 2, a substitution of E466H of SEQ ID NO:
2, a substitution of C477Q of SEQ ID NO: 2, a substitution of C477H of SEQ ID NO:
2, a substitution of C479A of SEQ ID NO: 2, a substitution of D659H of SEQ ID NO:
2, a substitution of T806V of SEQ ID NO: 2, a substitution of K8085 of SEQ ID NO:
2, an insertion of AS at position 797 of SEQ ID NO: 2, a substitution of V959M of SEQ ID NO:
2, a substitution of K975Q of SEQ ID NO: 2, a substitution of W974G of SEQ ID NO:
2, a substitution of A708Q of SEQ ID NO: 2, a substitution of V711K of SEQ ID NO:
2, a substitution of D733T of SEQ ID NO: 2, a substitution of L742W of SEQ ID NO:
2, a substitution of V747K of SEQ ID NO: 2, a substitution of F755M of SEQ ID NO:
2, a substitution of M771A of SEQ ID NO: 2, a substitution of M771Q of SEQ ID NO:
2, a substitution of W782Q of SEQ ID NO: 2, a substitution of G791F, of SEQ ID NO:
2 a substitution of L792D of SEQ ID NO: 2, a substitution of L792K of SEQ ID NO:
2, a substitution of P793Q of SEQ ID NO: 2, a substitution of P793G of SEQ ID NO:
2, a substitution of Q804A of SEQ ID NO: 2, a substitution of Y966N of SEQ ID NO:
2, a substitution of Y723N of SEQ ID NO: 2, a substitution of Y857R of SEQ ID NO:
2, a substitution of 5890R of SEQ ID NO: 2, a substitution of 5932M of SEQ ID NO:
2, a substitution of L897M of SEQ ID NO: 2, a substitution of R624G of SEQ ID NO:
2, a substitution of 5603G of SEQ ID NO: 2, a substitution of N737S of SEQ ID NO:
2, a substitution of L307K of SEQ ID NO: 2, a substitution of I658V of SEQ ID NO:
2, an insertion of PT at position 688 of SEQ ID NO: 2, an insertion of SA at position 794 of SEQ ID NO: 2, a substitution of 5877R of SEQ ID NO: 2, a substitution of N580T of SEQ ID NO:
2, a substitution of V335G of SEQ ID NO: 2, a substitution of T6205 of SEQ ID NO:
2, a substitution of W345G of SEQ ID NO: 2, a substitution of T2805 of SEQ ID NO:
2, a substitution of L406P of SEQ ID NO: 2, a substitution of A612D of SEQ ID NO:
2, a substitution of A751S of SEQ ID NO: 2, a substitution of E386R of SEQ ID NO:
2, a substitution of V351M of SEQ ID NO: 2, a substitution of K210N of SEQ ID NO:
2, a substitution of D40A of SEQ ID NO: 2, a substitution of E773G of SEQ ID NO: 2, a substitution of H207L of SEQ ID NO: 2, a substitution of T62A SEQ ID NO: 2, a substitution of T287P of SEQ ID NO: 2, a substitution of T832A of SEQ ID NO: 2, a substitution of A893S
of SEQ ID
NO: 2, an insertion of V at position 14 of SEQ ID NO: 2, an insertion of AG at position 13 of SEQ ID NO: 2, a substitution of R11V of SEQ ID NO: 2, a substitution of R12N
of SEQ ID NO:
2, a substitution of R13H of SEQ ID NO: 2, an insertion of Y at position 13 of SEQ ID NO: 2, a substitution of R12L of SEQ ID NO: 2, an insertion of Q at position 13 of SEQ
ID NO: 2, an substitution of V155 of SEQ ID NO: 2, an insertion of D at position 17 of SEQ
ID NO: 2 or a combination thereof.

[00240] In some embodiments, the CasX variant comprises at least one modification in the NTSB domain.

[00241] In some embodiments, the CasX variant comprises at least one modification in the TSL
domain. In some embodiments, the at least one modification in the TSL domain comprises an amino acid substitution of one or more of amino acids Y857, S890, or S932 of SEQ ID NO: 2.

[00242] In some embodiments, the CasX variant comprises at least one modification in the helical I domain. In some embodiments, the at least one modification in the helical I domain comprises an amino acid substitution of one or more of amino acids S219, L249, E259, Q252, E292, L307, or D318 of SEQ ID NO: 2.

[00243] In some embodiments, the CasX variant comprises at least one modification in the helical II domain. In some embodiments, the at least one modification in the helical II domain comprises an amino acid substitution of one or more of amino acids D361, L379, E385, E386, D387, F399, L404, R458, C477, or D489 of SEQ ID NO: 2.

[00244] In some embodiments, the CasX variant comprises at least one modification in the OBD domain. In some embodiments, the at least one modification in the OBD
comprises an amino acid substitution of one or more of amino acids F536, E552, T620, or 1658 of SEQ ID
NO: 2.

[00245] In some embodiments, the CasX variant comprises at least one modification in the RuvC DNA cleavage domain. In some embodiments, the at least one modification in the RuvC
DNA cleavage domain comprises an amino acid substitution of one or more of amino acids K682, G695, A708, V711, D732, A739, D733, L742, V747, F755, M771, M779, W782, A788, G791, L792, P793, Y797, M799, Q804, S819, or Y857 or a deletion of amino acid P793 of SEQ
ID NO: 2.

[00246] In some embodiments, the CasX variant comprises at least one modification compared to the reference CasX sequence of SEQ ID NO: 2 is selected from one or more of: (a) an amino acid substitution of L379R; (b) an amino acid substitution of A708K; (c) an amino acid substitution of T620P; (d) an amino acid substitution of E385P; (e) an amino acid substitution of Y857R; (f) an amino acid substitution of I658V; (g) an amino acid substitution of F399L; (h) an amino acid substitution of Q252K; (i) an amino acid substitution of L404K; and (j) an amino acid deletion of P793.

[00247] In some embodiments, a CasX variant protein comprises at least two amino acid changes to a reference CasX protein amino acid sequence. The at least two amino acid changes can be substitutions, insertions, or deletions of a reference CasX protein amino acid sequence, or any combination thereof. The substitutions, insertions or deletions can be any substitution, insertion or deletion in the sequence of a reference CasX protein described herein. In some embodiments, the changes are contiguous, non-contiguous, or a combination of contiguous and non-contiguous amino acid changes to a reference CasX protein sequence. In some embodiments, the reference CasX protein is SEQ ID NO: 2. In some embodiments, a CasX
variant protein comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95 or at least 100 amino acid changes to a reference CasX protein sequence. In some embodiments, a CasX variant protein comprises 1-50, 3-40, 5-30, 5-20, 5-15, 5-10, 10-50, 10-40, 10-30, 10-20, 15-50, 15-40, 15-30, 2-25, 2-24, 2-22, 2-23, 2-22, 2-21, 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-25, 3-24, 3-22, 3-23, 3-22, 3-21, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-25, 4-24, 4-22, 4-23, 4-22, 4-21, 4-20, 4-19, 4-18, 4-17, 4-16, 4-15, 4-14, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-25, 5-24, 5-22, 5-23, 5-22, 5-21, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7 or 5-6 amino acid changes to a reference CasX protein sequence. In some embodiments, a CasX variant protein comprises 15-20 changes to a reference CasX protein sequence. In some embodiments, a CasX variant protein comprises 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid changes to a reference CasX protein sequence. In some embodiments, the at least two amino acid changes to the sequence of a reference CasX variant protein are selected from the group consisting of: a substitution of Y789T of SEQ ID NO: 2, a deletion of P793 of SEQ ID NO: 2, a substitution of Y789D of SEQ ID NO: 2, a substitution of T725 of SEQ ID NO: 2, a substitution of I546V of SEQ ID NO: 2, a substitution of E552A of SEQ ID NO: 2, a substitution of A636D of SEQ ID NO: 2, a substitution of F5365 of SEQ ID
NO:2, a substitution of A708K of SEQ ID NO: 2, a substitution of Y797L of SEQ
ID NO: 2, a substitution of L792G SEQ ID NO: 2, a substitution of A739V of SEQ ID NO: 2, a substitution of G791M of SEQ ID NO: 2, an insertion of A at position 661of SEQ ID NO: 2, a substitution of A788W of SEQ ID NO: 2, a substitution of K390R of SEQ ID NO: 2, a substitution of A75 is of SEQ ID NO: 2, a substitution of E385A of SEQ ID NO: 2, an insertion of P at position 696 of SEQ ID NO: 2, an insertion of M at position 773 of SEQ ID NO: 2, a substitution of G695H of SEQ ID NO: 2, an insertion of AS at position 793 of SEQ ID NO: 2, an insertion of AS at position 795 of SEQ ID NO: 2, a substitution of C477R of SEQ ID NO: 2, a substitution of C477K of SEQ ID NO: 2, a substitution of C479A of SEQ ID NO: 2, a substitution of C479L of SEQ ID NO: 2, a substitution of I55F of SEQ ID NO: 2, a substitution of K21OR
of SEQ ID NO:
2, a substitution of C2335 of SEQ ID NO: 2, a substitution of D23 1N of SEQ ID
NO: 2, a substitution of Q338E of SEQ ID NO: 2, a substitution of Q338R of SEQ ID NO:
2, a substitution of L379R of SEQ ID NO: 2, a substitution of K390R of SEQ ID NO:
2, a substitution of L481Q of SEQ ID NO: 2, a substitution of F495S of SEQ ID NO:2, a substitution of D600N of SEQ ID NO: 2, a substitution of T886K of SEQ ID NO: 2, a substitution of A739V
of SEQ ID NO: 2, a substitution of K460N of SEQ ID NO: 2, a substitution of I199F of SEQ ID
NO: 2, a substitution of G492P of SEQ ID NO: 2, a substitution of T1531 of SEQ
ID NO: 2, a substitution of R591I of SEQ ID NO: 2, an insertion of AS at position 795 of SEQ ID NO: 2, an insertion of AS at position 796 of SEQ ID NO:2, an insertion of L at position 889 of SEQ ID
NO: 2, a substitution of E121D of SEQ ID NO: 2, a substitution of S270W of SEQ
ID NO: 2, a substitution of E712Q of SEQ ID NO: 2, a substitution of K942Q of SEQ ID NO:
2, a substitution of E552K of SEQ ID NO:2, a substitution of K25Q of SEQ ID NO: 2, a substitution of N47D of SEQ ID NO: 2, an insertion of T at position 696 of SEQ ID NO: 2, a substitution of L685I of SEQ ID NO: 2, a substitution of N880D of SEQ ID NO: 2, a substitution of Q102R of SEQ ID NO: 2, a substitution of M734K of SEQ ID NO: 2, a substitution of A7245 of SEQ ID
NO: 2, a substitution of T704K of SEQ ID NO: 2, a substitution of P224K of SEQ
ID NO: 2, a substitution of K25R of SEQ ID NO: 2, a substitution of M29E of SEQ ID NO: 2, a substitution of H152D of SEQ ID NO: 2, a substitution of 5219R of SEQ ID NO: 2, a substitution of E475K
of SEQ ID NO: 2, a substitution of G226R of SEQ ID NO: 2, a substitution of A377K of SEQ
ID NO: 2, a substitution of E480K of SEQ ID NO: 2, a substitution of K416E of SEQ ID NO: 2, a substitution of H164R of SEQ ID NO: 2, a substitution of K767R of SEQ ID NO:
2, a substitution of I7F of SEQ ID NO: 2, a substitution of M29R of SEQ ID NO: 2, a substitution of H435R of SEQ ID NO: 2, a substitution of E385Q of SEQ ID NO: 2, a substitution of E385K of SEQ ID NO: 2, a substitution of I279F of SEQ ID NO: 2, a substitution of D4895 of SEQ ID
NO: 2, a substitution of D732N of SEQ ID NO: 2, a substitution of A739T of SEQ
ID NO: 2, a substitution of W885R of SEQ ID NO: 2, a substitution of E53K of SEQ ID NO: 2, a substitution of A238T of SEQ ID NO: 2, a substitution of P283Q of SEQ ID NO:
2, a substitution of E292K of SEQ ID NO: 2, a substitution of Q628E of SEQ ID NO:
2, a substitution of R388Q of SEQ ID NO: 2, a substitution of G791M of SEQ ID NO:
2, a substitution of L792K of SEQ ID NO: 2, a substitution of L792E of SEQ ID NO:
2, a substitution of M779N of SEQ ID NO: 2, a substitution of G27D of SEQ ID NO: 2, a substitution of K955R of SEQ ID NO: 2, a substitution of 5867R of SEQ ID NO:
2, a substitution of R693I of SEQ ID NO: 2, a substitution of F189Y of SEQ ID NO:
2, a substitution of V635M of SEQ ID NO: 2, a substitution of F399L of SEQ ID NO: 2, a substitution of E498K
of SEQ ID NO: 2, a substitution of E386R of SEQ ID NO: 2, a substitution of V254G of SEQ
ID NO: 2, a substitution of P793S of SEQ ID NO: 2, a substitution of K188E of SEQ ID NO: 2, a substitution of QT945KI of SEQ ID NO: 2, a substitution of T620P of SEQ ID
NO: 2, a substitution of T946P of SEQ ID NO: 2, a substitution of TT949PP of SEQ ID NO:
2, a substitution of N952T of SEQ ID NO: 2, a substitution of K682E of SEQ ID NO:
2, a substitution of K975R of SEQ ID NO: 2, a substitution of L212P of SEQ ID NO:
2, a substitution of E292R of SEQ ID NO: 2, a substitution of 1303K of SEQ ID NO:
2, a substitution of C349E of SEQ ID NO: 2, a substitution of E385P of SEQ ID NO:
2, a substitution of E386N of SEQ ID NO: 2, a substitution of D387K of SEQ ID NO:
2, a substitution of L404K of SEQ ID NO: 2, a substitution of E466H of SEQ ID NO:
2, a substitution of C477Q of SEQ ID NO: 2, a substitution of C477H of SEQ ID NO:
2, a substitution of C479A of SEQ ID NO: 2, a substitution of D659H of SEQ ID NO:
2, a substitution of T806V of SEQ ID NO: 2, a substitution of K8085 of SEQ ID NO:
2, an insertion of AS at position 797 of SEQ ID NO: 2, a substitution of V959M of SEQ ID NO:
2, a substitution of K975Q of SEQ ID NO: 2, a substitution of W974G of SEQ ID NO:
2, a substitution of A708Q of SEQ ID NO: 2, a substitution of V711K of SEQ ID NO:
2, a substitution of D733T of SEQ ID NO: 2, a substitution of L742W of SEQ ID NO:
2, a substitution of V747K of SEQ ID NO: 2, a substitution of F755M of SEQ ID NO:
2, a substitution of M771A of SEQ ID NO: 2, a substitution of M771Q of SEQ ID NO:
2, a substitution of W782Q of SEQ ID NO: 2, a substitution of G791F, of SEQ ID NO:
2 a substitution of L792D of SEQ ID NO: 2, a substitution of L792K of SEQ ID NO:
2, a substitution of P793Q of SEQ ID NO: 2, a substitution of P793G of SEQ ID NO:
2, a substitution of Q804A of SEQ ID NO: 2, a substitution of Y966N of SEQ ID NO:
2, a substitution of Y723N of SEQ ID NO: 2, a substitution of Y857R of SEQ ID NO:
2, a substitution of 5890R of SEQ ID NO: 2, a substitution of 5932M of SEQ ID NO:
2, a substitution of L897M of SEQ ID NO: 2, a substitution of R624G of SEQ ID NO:
2, a substitution of 5603G of SEQ ID NO: 2, a substitution of N737S of SEQ ID NO:
2, a substitution of L307K of SEQ ID NO: 2, a substitution of I658V of SEQ ID NO:
2, an insertion of PT at position 688 of SEQ ID NO: 2, an insertion of SA at position 794 of SEQ ID NO: 2, a substitution of 5877R of SEQ ID NO: 2, a substitution of N580T of SEQ ID NO:
2, a substitution of V335G of SEQ ID NO: 2, a substitution of T6205 of SEQ ID NO:
2, a substitution of W345G of SEQ ID NO: 2, a substitution of T2805 of SEQ ID NO:
2, a substitution of L406P of SEQ ID NO: 2, a substitution of A612D of SEQ ID NO:
2, a substitution of A751S of SEQ ID NO: 2, a substitution of E386R of SEQ ID NO:
2, a substitution of V351M of SEQ ID NO: 2, a substitution of K210N of SEQ ID NO:
2, a substitution of D40A of SEQ ID NO: 2, a substitution of E773G of SEQ ID NO: 2, a substitution of H207L of SEQ ID NO: 2, a substitution of T62A SEQ ID NO: 2, a substitution of T287P of SEQ ID NO: 2, a substitution of T832A of SEQ ID NO: 2, a substitution of A893S
of SEQ ID
NO: 2, an insertion of V at position 14 of SEQ ID NO: 2, an insertion of AG at position 13 of SEQ ID NO: 2, a substitution of R11V of SEQ ID NO: 2, a substitution of R12N
of SEQ ID NO:
2, a substitution of R13H of SEQ ID NO: 2, an insertion of Y at position 13 of SEQ ID NO: 2, a substitution of R12L of SEQ ID NO: 2, an insertion of Q at position 13 of SEQ
ID NO: 2, an substitution of V155 of SEQ ID NO: 2 and an insertion of D at position 17 of SEQ ID NO: 2. In some embodiments, the at least two amino acid changes to a reference CasX
protein are selected from the amino acid changes disclosed in the sequences of Table 3. In some embodiments, a CasX variant comprises any combination of the foregoing embodiments of this paragraph.

[00248] In some embodiments, a CasX variant protein comprises more than one substitution, insertion and/or deletion of a reference CasX protein amino acid sequence. In some embodiments, the reference CasX protein comprises or consists essentially of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of 5794R and a substitution of Y797L of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of K416E and a substitution of A708K of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of A708K and a deletion of P793 of SEQ ID NO:
2. In some embodiments, a CasX variant protein comprises a deletion of P793 and an insertion of AS at position 795 SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of Q367K and a substitution of I425S of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of A708K, a deletion of P
position 793 and a substitution A793V of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of Q338R and a substitution of A339E of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of Q338R and a substitution of A339K of SEQ ID
NO: 2. In some embodiments, a CasX variant protein comprises a substitution of 5507G and a substitution of G508R of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of A708K and a deletion of P
at position 793 of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of C477K, a substitution of A708K and a deletion of P at position 793 of SEQ ID
NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K and a deletion of P at position of 793 of SEQ
ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a substitution A739V of SEQ ID NO:
2. In some embodiments, a CasX variant protein comprises a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of A739V of SEQ ID
NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of A739V of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a substitution of M779N of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a substitution of M771N of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of 708K, a deletion of P at position 793 and a substitution of D4895 of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a substitution of A739T of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a substitution of D732N of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a substitution of G791M of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of 708K, a deletion of P at position 793 and a substitution of Y797L of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of M779N of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of M771N of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of D4895 of SEQ
ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of A739T of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of D732N of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of G791M of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of Y797L of SEQ
ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of T620P of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of A708K, a deletion of P at position 793 and a substitution of E3 86S of SEQ ID
NO: 2. In some embodiments, a CasX variant protein comprises a substitution of E386R, a substitution of F399L
and a deletion of P at position 793 of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of R581I and A739V of SEQ ID NO: 2. In some embodiments, a CasX variant comprises any combination of the foregoing embodiments of this paragraph.

[00249] In some embodiments, a CasX variant protein comprises more than one substitution, insertion and/or deletion of a reference CasX protein amino acid sequence. In some embodiments, a CasX variant protein comprises a substitution of A708K, a deletion of P at position 793 and a substitution of A739V of SEQ ID NO: 2. In some embodiments, a CasX
variant protein comprises a substitution of L379R, a substitution of A708K and a deletion of P at position 793 of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of C477K, a substitution of A708K and a deletion of P at position 793 of SEQ ID
NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K and a deletion of P at position 793 of SEQ ID
NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a substitution of A739V of SEQ ID
NO: 2. In some embodiments, a CasX variant protein comprises a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of A739 of SEQ ID NO:
2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of A739V of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of T620P of SEQ ID NO: 2. In some embodiments, a CasX
variant protein comprises a substitution of M771A of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of A708K, a deletion of P
at position 793 and a substitution of D732N of SEQ ID NO: 2. In some embodiments, a CasX
variant comprises any combination of the foregoing embodiments of this paragraph.

[00250] In some embodiments, a CasX variant protein comprises a substitution of W782Q of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of M771Q of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of R458I and a substitution of A739V of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of A708K, a deletion of P
at position 793 and a substitution of M771N of SEQ ID NO: 2. In some embodiments, a CasX
variant protein comprises a substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a substitution of A739T of SEQ ID NO: 2. In some embodiments, a CasX
variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of D4895 of SEQ ID
NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of D732N of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of V711K of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of Y797L of SEQ ID NO: 2. In some embodiments, a CasX
variant protein comprises a substitution of L379R, a substitution of A708K and a deletion of P at position 793 of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of M771N of SEQ ID NO: 2. In some embodiments, a CasX

variant protein comprises a substitution of A708K, a substitution of P at position 793 and a substitution of E386S of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of C477K, a substitution of A708K and a deletion of P at position 793 of SEQ ID NO: 2. In some embodiments, a CasX
variant protein comprises a substitution of L792D of SEQ ID NO: 2. In some embodiments, a CasX
variant protein comprises a substitution of G791F of SEQ ID NO: 2. In some embodiments, a CasX
variant protein comprises a substitution of A708K, a deletion of P at position 793 and a substitution of A739V of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a substitution of A739V of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of C477K, a substitution of A708K and a substitution of P at position 793 of SEQ ID NO: 2. In some embodiments, a CasX variant protein comprises a substitution of L249I and a substitution of M771N of SEQ ID NO: 2. In some embodiments, a CasX
variant protein comprises a substitution of V747K of SEQ ID NO: 2. In some embodiments, a CasX
variant protein comprises a substitution of L379R, a substitution of C477, a substitution of A708K, a deletion of P at position 793 and a substitution of M779N of SEQ ID
NO: 2. In some embodiments, a CasX variant protein comprises a substitution of F755M. In some embodiments, a CasX variant comprises any combination of the foregoing embodiments of this paragraph.

[00251] In some embodiments, a CasX variant protein comprises at least one modification compared to the reference CasX sequence of SEQ ID NO: 2, wherein the at least one modification is selected from one or more of: an amino acid substitution of L379R; an amino acid substitution of A708K; an amino acid substitution of T620P; an amino acid substitution of E385P; an amino acid substitution of Y857R; an amino acid substitution of I658V; an amino acid substitution of F399L; an amino acid substitution of Q252K; an amino acid substitution of L404K; and an amino acid deletion of [P793]. In other embodiments, a CasX
variant protein comprises any combination of the foregoing substitutions or deletions compared to the reference CasX sequence of SEQ ID NO: 2. In other embodiments, the CasX variant protein can, in addition to the foregoing substitutions or deletions, further comprise a substitution of an NTSB
and/or a helical lb domain from the reference CasX of SEQ ID NO: 1.

[00252] In some embodiments, a CasX variant comprises any one of SEQ ID NOS:
247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4412-4415. In some embodiments, a CasX

variant comprises any one of SEQ ID NOS: 247-337, 3498-3501, 3505-3520, 3540-3549 and 4412-4415. In some embodiments, a CasX variant comprises any one of SEQ ID
NOS: 3498-3501, 3505-3520 and 3540-3549.

[00253] In some embodiments, a CasX variant comprises one or modifications to any one of SEQ ID NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4412-4415.
In some embodiments, a CasX variant comprises one or modifications to any one of SEQ
ID NOS: 247-337, 3498-3501, 3505-3520, 3540-3549 and 4412-4415. In some embodiments, a CasX variant comprises one or modifications to any one of SEQ ID NOS: 3498-3501, 3505-3520 and 3540-3549.

[00254] In some embodiments, the CasX variant protein comprises between 400 and 2000 amino acids, between 500 and 1500 amino acids, between 700 and 1200 amino acids, between 800 and 1100 amino acids or between 900 and 1000 amino acids.

[00255] In some embodiments, the CasX variant protein comprises one or more modifications in a region of non-contiguous residues that form a channel in which gNA:target DNA
complexing occurs. In some embodiments, the CasX variant protein comprises one or more modifications comprising a region of non-contiguous residues that form an interface which binds with the gNA. For example, in some embodiments of a reference CasX protein, the helical I, helical II and OBD domains all contact or are in proximity to the gNA:target DNA complex, and one or more modifications to non-contiguous residues within any of these domains may improve function of the CasX variant protein.

[00256] In some embodiments, the CasX variant protein comprises one or more modifications in a region of non-contiguous residues that form a channel which binds with the non-target strand DNA. For example, a CasX variant protein can comprise one or more modifications to non-contiguous residues of the NTSBD. In some embodiments, the CasX variant protein comprises one or more modifications in a region of non-contiguous residues that form an interface which binds with the PAM. For example, a CasX variant protein can comprise one or more modifications to non-contiguous residues of the helical I domain or OBD.
In some embodiments, the CasX variant protein comprises one or more modifications comprising a region of non-contiguous surface-exposed residues. As used herein, "surface-exposed residues"
refers to amino acids on the surface of the CasX protein, or amino acids in which at least a portion of the amino acid, such as the backbone or a part of the side chain is on the surface of the protein. Surface exposed residues of cellular proteins such as CasX, which are exposed to an aqueous intracellular environment, are frequently selected from positively charged hydrophilic amino acids, for example arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine. Thus, for example, in some embodiments of the variants provided herein, a region of surface exposed residues comprises one or more insertions, deletions, or substitutions compared to a reference CasX protein. In some embodiments, one or more positively charged residues are substituted for one or more other positively charged residues, or negatively charged residues, or uncharged residues, or any combinations thereof. In some embodiments, one or more amino acids residues for substitution are near bound nucleic acid, for example residues in the RuvC domain or helical I domain that contact target DNA, or residues in the OBD or helical II domain that bind the gNA, can be substituted for one or more positively charged or polar amino acids.

[00257] In some embodiments, the CasX variant protein comprises one or more modifications in a region of non-contiguous residues that form a core through hydrophobic packing in a domain of the reference CasX protein. Without wishing to be bound by any theory, regions that form cores through hydrophobic packing are rich in hydrophobic amino acids such as valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, and cysteine. For example, in some reference CasX proteins, RuvC domains comprise a hydrophobic pocket adjacent to the active site. In some embodiments, between 2 to 15 residues of the region are charged, polar, or base-stacking. Charged amino acids (sometimes referred to herein as residues) may include, for example, arginine, lysine, aspartic acid, and glutamic acid, and the side chains of these amino acids may form salt bridges provided a bridge partner is also present (see FIGS. 14). Polar amino acids may include, for example, glutamine, asparagine, histidine, serine, threonine, tyrosine, and cysteine. Polar amino acids can, in some embodiments, form hydrogen bonds as proton donors or acceptors, depending on the identity of their side chains. As used herein, "base-stacking"
includes the interaction of aromatic side chains of an amino acid residue (such as tryptophan, tyrosine, phenylalanine, or histidine) with stacked nucleotide bases in a nucleic acid. Any modification to a region of non-contiguous amino acids that are in close spatial proximity to form a functional part of the CasX variant protein is envisaged as within the scope of the disclosure.
i. CasX Variant Proteins with Domains from Multiple Source Proteins

[00258] In certain embodiments, the disclosure provides a chimeric CasX
protein comprising protein domains from two or more different CasX proteins, such as two or more naturally occurring CasX proteins, or two or more CasX variant protein sequences as described herein.
As used herein, a "chimeric CasX protein" refers to a CasX containing at least two domains isolated or derived from different sources, such as two naturally occurring proteins, which may, in some embodiments, be isolated from different species. For example, in some embodiments, a chimeric CasX protein comprises a first domain from a first CasX protein and a second domain from a second, different CasX protein. In some embodiments, the first domain can be selected from the group consisting of the NTSB, TSL, helical I, helical II, OBD and RuvC domains. In some embodiments, the second domain is selected from the group consisting of the NTSB, TSL, helical I, helical II, OBD and RuvC domains with the second domain being different from the foregoing first domain. For example, a chimeric CasX protein may comprise an NTSB, TSL, helical I, helical II, OBD domains from a CasX protein of SEQ ID NO: 2, and a RuvC domain from a CasX protein of SEQ ID NO: 1, or vice versa. As a further example, a chimeric CasX
protein may comprise an NTSB, TSL, helical II, OBD and RuvC domain from CasX
protein of SEQ ID NO: 2, and a helical I domain from a CasX protein of SEQ ID NO: 1, or vice versa.
Thus, in certain embodiments, a chimeric CasX protein may comprise an NTSB, TSL, helical II, OBD and RuvC domain from a first CasX protein, and a helical I domain from a second CasX
protein. In some embodiments of the chimeric CasX proteins, the domains of the first CasX
protein are derived from the sequences of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID
NO: 3, and the domains of the second CasX protein are derived from the sequences of SEQ
ID NO: 1, SEQ
ID NO: 2 or SEQ ID NO: 3, and the first and second CasX proteins are not the same. In some embodiments, domains of the first CasX protein comprise sequences derived from SEQ ID NO:
1 and domains of the second CasX protein comprise sequences derived from SEQ
ID NO: 2. In some embodiments, domains of the first CasX protein comprise sequences derived from SEQ
ID NO: 1 and domains of the second CasX protein comprise sequences derived from SEQ ID
NO: 3. In some embodiments, domains of the first CasX protein comprise sequences derived from SEQ ID NO: 2 and domains of the second CasX protein comprise sequences derived from SEQ ID NO: 3. In some embodiments, the CasX variant is selected of group consisting of CasX
variants with sequences of SEQ ID NO: 328, SEQ ID NO: 3540, SEQ ID NO: 4413, SEQ ID
NO: 4414, SEQ ID NO: 4415, SEQ ID NO: 329, SEQ ID NO: 3541, SEQ ID NO: 330, SEQ ID
NO: 3542, SEQ ID NO: 331, SEQ ID NO: 3543, SEQ ID NO: 332, SEQ ID NO: 3544, SEQ ID
NO: 333, SEQ ID NO: 3545, SEQ ID NO: 334, SEQ ID NO: 3546, SEQ ID NO: 335, SEQ
ID
NO: 3547, SEQ ID NO: 336 and SEQ ID NO: 3548. In some embodiments, the CasX
variant comprises one or more additional modifications to any one of SEQ ID NO: 328, SEQ ID NO:
3540, SEQ ID NO: 4413, SEQ ID NO: 4414, SEQ ID NO: 4415, SEQ ID NO: 329, SEQ
ID NO:
3541, SEQ ID NO: 330, SEQ ID NO: 3542, SEQ ID NO: 331, SEQ ID NO: 3543, SEQ ID
NO:
332, SEQ ID NO: 3544, SEQ ID NO: 333, SEQ ID NO: 3545, SEQ ID NO: 334, SEQ ID
NO:
3546, SEQ ID NO: 335, SEQ ID NO: 3547, SEQ ID NO: 336 or SEQ ID NO: 3548. In some embodiments, the one or more additional modifications comprises an insertion, substitution or deletion as described herein.

[00259] In some embodiments, a CasX variant protein comprises at least one chimeric domain comprising a first part from a first CasX protein and a second part from a second, different CasX
protein. As used herein, a "chimeric domain" refers to a domain containing at least two parts isolated or derived from different sources, such as two naturally occurring proteins or portions of domains from two reference CasX proteins. The at least one chimeric domain can be any of the NTSB, TSL, helical I, helical II, OBD or RuvC domains as described herein. In some embodiments, the first portion of a CasX domain comprises a sequence of SEQ ID
NO: 1 and the second portion of a CasX domain comprises a sequence of SEQ ID NO: 2. In some embodiments, the first portion of the CasX domain comprises a sequence of SEQ
ID NO: 1 and the second portion of the CasX domain comprises a sequence of SEQ ID NO: 3. In some embodiments, the first portion of the CasX domain comprises a sequence of SEQ
ID NO: 2 and the second portion of the CasX domain comprises a sequence of SEQ ID NO: 3. In some embodiments, the at least one chimeric domain comprises a chimeric RuvC
domain. As an example of the foregoing, the chimeric RuvC domain comprises amino acids 661 to 824 of SEQ
ID NO: 1 and amino acids 922 to 978 of SEQ ID NO: 2. As an alternative example of the foregoing, a chimeric RuvC domain comprises amino acids 648 to 812 of SEQ ID
NO: 2 and amino acids 935 to 986 of SEQ ID NO: 1. In some embodiments, a CasX protein comprises a first domain from a first CasX protein and a second domain from a second CasX
protein, and at least one chimeric domain comprising at least two parts isolated from different CasX proteins using the approach of the embodiments described in this paragraph. In the foregoing embodiments, the chimeric CasX proteins having domains or portions of domains derived from SEQ ID NOS: 1, 2 and 3, can further comprise amino acid insertions, deletions, or substitutions of any of the embodiments disclosed herein.

[00260] In some embodiments, a CasX variant protein comprises a sequence set forth in Tables 3, 8, 9, 10 or 12. In other embodiments, a CasX variant protein comprises a sequence at least 60% identical, at least 65% identical, at least 70% identical, at least 75%
identical, at least 80%
identical, at least 81% identical, at least 82% identical, at least 83%
identical, at least 84%
identical, at least 85% identical, at least 86% identical, at least 86%
identical, at least 87%
identical, at least 88% identical, at least 89% identical, at least 89%
identical, at least 90%
identical, at least 91% identical, at least 92% identical, at least 93%
identical, at least 94%
identical, at least 95% identical, at least 96% identical, at least 97%
identical, at least 98%
identical, at least 99% identical, at least 99.5% identical to a sequence set forth in Tables 3, 8, 9, or 12. In other embodiments, a CasX variant protein comprises a sequence set forth in Table 3, and further comprises one or more NLS disclosed herein on either the N-terminus, the C-terminus, or both. It will be understood that in some cases, the N-terminal methionine of the CasX variants of the Tables is removed from the expressed CasX variant during post-translational modification.
Table 3: CasX Variant Sequences Description*
Amino Acid Sequence TSL, Helical I, Helical II, OBD and RuvC domains from SEQ ID NO: 2 and an NTSB
SEQ ID NO: 247 domain from SEQ ID NO: 1 NTSB, Helical I, Helical II, OBD and RuvC domains from SEQ ID NO: 2 and a TSL
SEQ ID NO: 248 domain from SEQ ID NO: 1.
TSL, Helical I, Helical II, OBD and RuvC domains from SEQ ID NO: 1 and an NTSB
SEQ ID NO: 249 domain from SEQ ID NO: 2 NTSB, Helical I, Helical II, OBD and RuvC domains from SEQ ID NO: 1 and an TSL
SEQ ID NO: 250 domain from SEQ ID NO: 2.
NTSB, TSL, Helical I, Helical II and OBD domains SEQ ID NO: 2 and an exogenous SEQ ID NO: 251 RuvC domain or a portion thereof from a second CasX protein.
No description SEQ ID NO: 252 NTSB, TSL, Helical II, OBD and RuvC domains from SEQ ID NO: 2 and a Helical I
SEQ ID NO: 253 domain from SEQ ID NO: 1 NTSB, TSL, Helical I, OBD and RuvC domains from SEQ ID NO: 2 and a Helical II
SEQ ID NO: 254 domain from SEQ ID NO: 1 NTSB, TSL, Helical I, Helical II and RuvC domains from a first CasX protein and an SEQ ID NO: 255 exogenous OBD or a part thereof from a second CasX protein No description SEQ ID NO: 256 No description SEQ ID NO: 257 substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of SEQ ID NO: 258 P at position 793 and a substitution of T620P of SEQ ID NO: 2 Description*
Amino Acid Sequence substitution of M771A of SEQ ID NO: 2. SEQ ID NO: 259 substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a SEQ ID NO: 260 substitution of D732N of SEQ ID NO: 2.
substitution of W782Q of SEQ ID NO: 2. SEQ ID NO: 261 substitution of M771Q of SEQ ID NO: 2 SEQ ID NO: 262 substitution of R458I and a substitution of A739V of SEQ ID NO: 2. SEQ ID
NO: 263 L379R, a substitution of A708K, a deletion of P at position 793 and a substitution of SEQ ID NO: 264 M771N of SEQ ID NO: 2 substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a SEQ ID NO: 265 substitution of A739T of SEQ ID NO: 2 substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of SEQ ID NO: 266 P at position 793 and a substitution of D4895 of SEQ ID NO: 2.
substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of SEQ ID NO: 267 P at position 793 and a substitution of D732N of SEQ ID NO: 2.
substitution of V711K of SEQ ID NO: 2. SEQ ID NO: 268 substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of SEQ ID NO: 269 P at position 793 and a substitution of Y797L of SEQ ID NO: 2.
119, substitution of L379R, a substitution of A708K and a deletion of P at position 793 SEQ ID NO: 270 of SEQ ID NO: 2.
substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of SEQ ID NO: 271 P at position 793 and a substitution of M771N of SEQ ID NO: 2.
substitution of A708K, a deletion of P at position 793 and a substitution of E386S of SEQ ID NO: 272 SEQ ID NO: 2.
substitution of L379R, a substitution of C477K, a substitution of A708K and a deletion SEQ ID NO: 273 of P at position 793 of SEQ ID NO: 2.
substitution of L792D of SEQ ID NO: 2. SEQ ID NO: 274 substitution of G791F of SEQ ID NO: 2. SEQ ID NO: 275 substitution of A708K, a deletion of P at position 793 and a substitution of A739V of SEQ ID NO: 276 SEQ ID NO: 2.
substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a (SEQ ID NO: 277 substitution of A739V of SEQ ID NO: 2.
substitution of C477K, a substitution of A708K and a deletion of P at position 793 of SEQ ID NO: 278 SEQ ID NO: 2.
substitution of L249I and a substitution of M77 1N of SEQ ID NO: 2. SEQ ID
NO: 279 substitution of V747K of SEQ ID NO: 2. SEQ ID NO: 280 substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of SEQ ID NO: 281 Description*
Amino Acid Sequence P at position 793 and a substitution of M779N of SEQ ID NO: 2.
L379R, F755M
SEQ ID NO: 282 429, L379R, A708K, P793_, Y857R SEQ ID NO: 283 430, L379R, A708K, P793_, Y857R, I658V SEQ ID NO: 284 431, L379R, A708K, P793_, Y857R, I658V, E386N SEQ ID NO: 285 432, L379R, A708K, P793_, Y857R, I658V, L404K SEQ ID NO: 286 433, L379R, A708K, P793_, Y857R, I658V, AV192 SEQ ID NO: 287 434, L379R, A708K, P793_, Y857R, I658V, L404K, E386N SEQ ID NO: 288 435, L379R, A708K, P793_, Y857R, I658V, F399L SEQ ID NO: 289 436, L379R, A708K, P793_, Y857R, I658V, F399L, E386N SEQ ID NO: 290 437, L379R, A708K, P793_, Y857R, I658V, F399L, C4775 SEQ ID NO: 291 438, L379R, A708K, P793_, Y857R, I658V, F399L, L404K SEQ ID NO: 292 439, L379R, A708K, P793_, Y857R, I658V, F399L, E386N, C4775, L404K SEQ ID
NO: 293 440, L379R, A708K, P793_, Y857R, I658V, F399L, Y797L SEQ ID NO: 294 441, L379R, A708K, P793_, Y857R, I658V, F399L, Y797L, E386N SEQ ID NO: 295 442, L379R, A708K, P793_, Y857R, I658V, F399L, Y797L, E386N, C4775, L404K SEQ
ID NO: 296 443, L379R, A708K, P793_, Y857R, I658V, Y797L SEQ ID NO: 297 444, L379R, A708K, P793_, Y857R, I658V, Y797L, L404K SEQ ID NO: 298 445, L379R, A708K, P793_, Y857R, I658V, Y797L, E386N SEQ ID NO: 299 446, L379R, A708K, P793_, Y857R, I658V, Y797L, E386N, C4775, L404K SEQ ID
NO: 300 447, L379R, A708K, P793_, Y857R, E386N SEQ ID NO: 301 448, L379R, A708K, P793_, Y857R, E386N, L404K SEQ ID NO: 302 449, L379R, A708K, P793_, D732N, E385P, Y857R SEQ ID NO: 303 450, L379R, A708K, P793_, D732N, E385P, Y857R, I658V SEQ ID NO: 304 451, L379R, A708K, P793_, D732N, E385P, Y857R, I658V, F399L SEQ ID NO: 305 452, L379R, A708K, P793_, D732N, E385P, Y857R, I658V, E386N SEQ ID NO: 306 453, L379R, A708K, P793_, D732N, E385P, Y857R, I658V, L404K SEQ ID NO: 307 454, L379R, A708K, P793_, T620P, E385P, Y857R, Q252K SEQ ID NO: 308 455, L379R, A708K, P793_, T620P, E385P, Y857R, I658V, Q252K SEQ ID NO: 309 456, L379R, A708K, P793_, T620P, E385P, Y857R, I658V, E386N, Q252K SEQ ID
NO: 310 Description*
Amino Acid Sequence 457, L379R, A708K, P793_, T620P, E385P, Y857R, I658V, F399L, Q252K SEQ ID
NO: 311 458, L379R, A708K, P793_, T620P, E385P, Y857R, I658V, L404K, Q252K
SEQ ID NO: 312 459, L379R, A708K, P793_, T620P, Y857R, I658V, E386N SEQ ID NO: 313 460, L379R, A708K, P793_, T620P, E385P, Q252K SEQ ID NO: 314 278 SEQ ID NO: 315 279 SEQ ID NO: 316 280 SEQ ID NO: 317 285 SEQ ID NO: 318 286 SEQ ID NO: 319 287 SEQ ID NO: 320 288 SEQ ID NO: 321 290 SEQ ID NO: 322 291 SEQ ID NO: 323 293 SEQ ID NO: 324 300 SEQ ID NO: 325 492 SEQ ID NO: 326 493 SEQ ID NO: 327 387, NTSB swap from SEQ ID NO: 1 SEQ ID NO: 328 395, Helical 1B swap from SEQ ID NO: 1 SEQ ID NO: 329 485, Helical 1B swap from SEQ ID NO: 1 SEQ ID NO: 330 486, Helical 1B swap from SEQ ID NO: 1 SEQ ID NO: 331 487, Helical 1B swap from SEQ ID NO: 1 SEQ ID NO: 332 488, NTSB and Helical 1B swap from SEQ ID NO: 1 SEQ ID NO: 333 489, NTSB and Helical 1B swap from SEQ ID NO: 1 SEQ ID NO: 334 490, NTSB and Helical 1B swap from SEQ ID NO: 1 SEQ ID NO: 335 491, NTSB and Helical 1B swap from SEQ ID NO: 1 SEQ ID NO: 336 494, NTSB swap from SEQ ID NO: 1 SEQ ID NO: 337 328, 5867G SEQ ID NO: 4412 388, L379R+A708K+ [P793] + X1 Helical2 swap SEQ ID NO: 4413 389, L379R+A708K+ [P793] + X1 RuvC1 swap SEQ ID NO: 4414 Description*
Amino Acid Sequence 390, L379R+A708K+ [P793] + X1 RuvC2 swap SEQ ID NO: 4415 * Strain indicated numerically; changes, where indicated, are relative to SEQ
ID NO: 2

[00261] In some embodiments, the CasX variant protein has one or more improved characteristics when compared to a reference CasX protein, for example a reference protein of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, an improved characteristic of the CasX variant is at least about 1.1 to about 100,000-fold improved relative to the reference protein. In some embodiments, an improved characteristic of the CasX variant is at least about 1.1 to about 10,000-fold improved, at least about 1.1 to about 1,000-fold improved, at least about 1.1 to about 500-fold improved, at least about 1.1 to about 400-fold improved, at least about 1.1 to about 300-fold improved, at least about 1.1 to about 200-fold improved, at least about 1.1 to about 100-fold improved, at least about 1.1 to about 50-fold improved, at least about 1.1 to about 40-fold improved, at least about 1.1 to about 30-fold improved, at least about 1.1 to about 20-fold improved, at least about 1.1 to about 10-fold improved, at least about 1.1 to about 9-fold improved, at least about 1.1 to about 8-fold improved, at least about 1.1 to about 7-fold improved, at least about 1.1 to about 6-fold improved, at least about 1.1 to about 5-fold improved, at least about 1.1 to about 4-fold improved, at least about 1.1 to about 3-fold improved, at least about 1.1 to about 2-fold improved, at least about 1.1 to about 1.5-fold improved, at least about 1.5 to about 3-fold improved, at least about 1.5 to about 4-fold improved, at least about 1.5 to about 5-fold improved, at least about 1.5 to about 10-fold improved, at least about 5 to about 10-fold improved, at least about 10 to about 20-fold improved, at least 10 to about 30-fold improved, at least 10 to about 50-fold improved or at least to about 100-fold improved than the reference CasX protein. In some embodiments, an improved characteristic of the CasX variant is at least about 10 to about 1000-fold improved relative to the reference CasX protein.

[00262] In some embodiments, the one or more improved characteristics of the CasX variant protein is at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 250, at least about 500, or at least about 1000, at least about 5,000, at least about 10,000, or at least about 100,000-fold improved relative to a reference CasX protein.
In some embodiments, an improved characteristics of the CasX variant protein is at least about 1.1, at least about 1.2, at least about 1.3, at least about 1.4, at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, at least about 2, at least about 2.1, at least about 2.2, at least about 2.3, at least about 2.4, at least about 2.5, at least about 2.6, at least about 2.7, at least about 2.8, at least about 2.9, at least about 3, at least about 3.5, at least about 4, at least about 4.5, at least about 5, at least about 5.5, at least about 6, at least about 6.5, at least about 7.0, at least about 7.5, at least about 8, at least about 8.5, at least about 9, at least about 9.5, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90 at least about 100, at least about 500, at least about 1,000, at least about 10,000, or at least about 100,000-fold improved relative to a reference CasX protein. In other cases, the one or more improved characteristics of the CasX variant is about 1.1 to 100,00-fold, about 1.1 to 10,00-fold, about 1.1 to 1,000-fold, about 1.1 to 500-fold, about 1.1 to 100-fold, about 1.1 to 50-fold, about 1.1 to 20-fold, about 10 to 100,00-fold, about to 10,00-fold, about 10 to 1,000-fold, about 10 to 500-fold, about 10 to 100-fold, about 10 to 50-fold, about 10 to 20-fold, about 2 to 70-fold, about 2 to 50-fold, about 2 to 30-fold, about 2 to 20-fold, about 2 to 10-fold, about 5 to 50-fold, about 5 to 30-fold, about 5 to 10-fold, about 100 to 100,00-fold, about 100 to 10,00-fold, about 100 to 1,000-fold, about 100 to 500-fold, about 500 to 100,00-fold, about 500 to 10,00-fold, about 500 to 1,000-fold, about 500 to 750-fold, about 1,000 to 100,00-fold, about 10,000 to 100,00-fold, about 20 to 500-fold, about 20 to 250-fold, about 20 to 200-fold, about 20 to 100-fold, about 20 to 50-fold, about 50 to 10,000-fold, about 50 to 1,000-fold, about 50 to 500-fold, about 50 to 200-fold, or about 50 to 100-fold, improved relative to the reference CasX of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ
ID NO: 3. In other cases, the one or more improved characteristics of the CasX variant is about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, 180-fold, 190-fold, 200-fold, 210-fold, 220-fold, 230-fold, 240-fold, 250-fold, 260-fold, 270-fold, 280-fold, 290-fold, 300-fold, 310-fold, 320-fold, 330-fold, 340-fold, 350-fold, 360-fold, 370-fold, 380-fold, 390-fold, 400-fold, 425-fold, 450-fold, 475-fold, or 500-fold or more improved relative to the reference CasX of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO:
3. Exemplary characteristics that can be improved in CasX variant proteins relative to the same characteristics in reference CasX proteins include, but are not limited to, improved folding of the variant, improved binding affinity to the gNA, improved binding affinity to the target DNA, improved ability to utilize a greater spectrum of PAM sequences in the editing and/or binding of target DNA, improved unwinding of the target DNA, increased editing activity, improved editing efficiency, improved editing specificity, increased activity of the nuclease, increased target strand loading for double strand cleavage, decreased target strand loading for single strand nicking, decreased off-target cleavage, improved binding of the non-target strand of DNA, improved protein stability, improved CasX:gNA RNA complex stability, improved protein solubility, improved CasX:gNA RNP complex solubility, improved protein yield, improved protein expression, and improved fusion characteristics. In some embodiments, the variant comprises at least one improved characteristic. In other embodiments, the variant comprises at least two improved characteristics. In further embodiments, the variant comprises at least three improved characteristics. In some embodiments, the variant comprises at least four improved characteristics. In still further embodiments, the variant comprises at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, or more improved characteristics. These improved characteristics are described in more detail below.
j. Protein Stability

[00263] In some embodiments, the disclosure provides a CasX variant protein with improved stability relative to a reference CasX protein. In some embodiments, improved stability of the CasX variant protein results in expression of a higher steady state of protein, which improves editing efficiency. In some embodiments, improved stability of the CasX
variant protein results in a larger fraction of CasX protein that remains folded in a functional conformation and improves editing efficiency or improves purifiability for manufacturing purposes. As used herein, a "functional conformation" refers to a CasX protein that is in a conformation where the protein is capable of binding a gNA and target DNA. In embodiments wherein the CasX variant does not carry one or more mutations rendering it catalytically dead, the CasX
variant is capable of cleaving, nicking, or otherwise modifying the target DNA. For example, a functional CasX
variant can, in some embodiments, be used for gene-editing, and a functional conformation refers to an "editing-competent" conformation. In some exemplary embodiments, including those embodiments where the CasX variant protein results in a larger fraction of CasX protein that remains folded in a functional conformation, a lower concentration of CasX variant is needed for applications such as gene editing compared to a reference CasX
protein. Thus, in some embodiments, the CasX variant with improved stability has improved efficiency compared to a reference CasX in one or more gene editing contexts.

[00264] In some embodiments, the disclosure provides a CasX variant protein having improved thermostability relative to a reference CasX protein. In some embodiments, the CasX variant protein has improved thermostability of the CasX variant protein at a particular temperature range. Without wishing to be bound by any theory, some reference CasX proteins natively function in organisms with niches in groundwater and sediment; thus, some reference CasX
proteins may have evolved to exhibit optimal function at lower or higher temperatures that may be desirable for certain applications. For example, one application of CasX
variant proteins is gene editing of mammalian cells, which is typically carried out at about 37 C.
In some embodiments, a CasX variant protein as described herein has improved thermostability compared to a reference CasX protein at a temperature of at least 16 C, at least 18 C, at least 20 C, at least 22 C, at least 24 C, at least 26 C, at least 28 C, at least 30 C, at least 32 C, at least 34 C, at least 35 C, at least 36 C, at least 37 C, at least 38 C, at least 39 C, at least 40 C, at least 41 C, at least 42 C, at least 44 C, at least 46 C, at least 48 C, at least 50 C, at least 52 C, or greater. In some embodiments, a CasX variant protein has improved thermostability and functionality compared to a reference CasX protein that results in improved gene editing functionality, such as mammalian gene editing applications, which may include human gene editing applications.

[00265] In some embodiments, the disclosure provides a CasX variant protein having improved stability of the CasX variant protein:gNA RNP complex relative to the reference CasX
protein:gNA complex such that the RNP remains in a functional form. Stability improvements can include increased thermostability, resistance to proteolytic degradation, enhanced pharmacolcinetic properties, stability across a range of pH conditions, salt conditions, and tonicity. Improved stability of the complex may, in some embodiments, lead to improved editing efficiency.

[00266] In some embodiments, the disclosure provides a CasX variant protein having improved thermostability of the CasX variant protein:gNA complex relative to the reference CasX
protein:gNA complex. In some embodiments, a CasX variant protein has improved thermostability relative to a reference CasX protein. In some embodiments, the CasX variant protein:gNA RNP complex has improved thermostability relative to a complex comprising a reference CasX protein at temperatures of at least 16 C, at least 18 C, at least 20 C, at least 22 C, at least 24 C, at least 26 C, at least 28 C, at least 30 C, at least 32 C, at least 34 C, at least 35 C, at least 36 C, at least 37 C, at least 38 C, at least 39 C, at least 40 C, at least 41 C, at least 42 C, at least 44 C, at least 46 C, at least 48 C, at least 50 C, at least 52 C, or greater.
In some embodiments, a CasX variant protein has improved thermostability of the CasX variant protein:gNA RNP complex compared to a reference CasX protein:gNA complex, which results in improved function for gene editing applications, such as mammalian gene editing applications, which may include human gene editing applications.

[00267] In some embodiments, the improved stability and/or thermostability of the CasX
variant protein comprises faster folding kinetics of the CasX variant protein relative to a reference CasX protein, slower unfolding kinetics of the CasX variant protein relative to a reference CasX protein, a larger free energy release upon folding of the CasX
variant protein relative to a reference CasX protein, a higher temperature at which 50% of the CasX variant protein is unfolded (Tm) relative to a reference CasX protein, or any combination thereof. These characteristics may be improved by a wide range of values; for example, at least 1.1, at least 1.5, at least 10, at least 50, at least 100, at least 500, at least 1,000, at least 5,000, or at least a 10,000-fold improved, as compared to a reference CasX protein. In some embodiments, improved thermostability of the CasX variant protein comprises a higher Tm of the CasX
variant protein relative to a reference CasX protein. In some embodiments, the Tm of the CasX
variant protein is between about 20 C to about 30 C, between about 30 C to about 40 C, between about 40 C
to about 50 C, between about 50 C to about 60 C, between about 60 C to about 70 C, between about 70 C to about 80 C, between about 80 C to about 90 C or between about 90 C to about 100 C. Thermal stability is determined by measuring the "melting temperature"
(T.), which is defined as the temperature at which half of the molecules are denatured.
Methods of measuring characteristics of protein stability such as Tm and the free energy of unfolding are known to persons of ordinary skill in the art, and can be measured using standard biochemical techniques in vitro. For example, Tm may be measured using Differential Scanning Calorimetry, a thermo-analytical technique in which the difference in the amount of heat required to increase the temperature of a sample and a reference is measured as a function of temperature (Chen et al (2003) Pharm Res 20:1952-60; Ghirlando et al (1999) Immunol Lett 68:47-52).
Alternatively, or in addition, CasX variant protein Tm may be measured using commercially available methods such as the ThermoFisher Protein Thermal Shift system. Alternatively, or in addition, circular dichroism may be used to measure the kinetics of folding and unfolding, as well as the Tm (Murray et al. (2002) J. Chromatogr Sci 40:343-9). Circular dichroism (CD) relies on the unequal absorption of left-handed and right-handed circularly polarized light by asymmetric molecules such as proteins. Certain structures of proteins, for example alpha-helices and beta-sheets, have characteristic CD spectra. Accordingly, in some embodiments, CD
may be used to determine the secondary structure of a CasX variant protein.

[00268] In some embodiments, improved stability and/or thermostability of the CasX variant protein comprises improved folding kinetics of the CasX variant protein relative to a reference CasX protein. In some embodiments, folding kinetics of the CasX variant protein are improved relative to a reference CasX protein by at least about 5, at least about 10, at least about 50, at least about 100, at least about 500, at least about 1,000, at least about 2,000, at least about 3,000, at least about 4,000, at least about 5,000, or at least about a 10,000-fold improvement. In some embodiments, folding kinetics of the CasX variant protein are improved relative to a reference CasX protein by at least about 1 kJ/mol, at least about 5 kJ/mol, at least about 10 kJ/mol, at least about 20 kJ/mol, at least about 30 kJ/mol, at least about 40 kJ/mol, at least about 50 kJ/mol, at least about 60 kJ/mol, at least about 70 kJ/mol, at least about 80 kJ/mol, at least about 90 kJ/mol, at least about 100 kJ/mol, at least about 150 kJ/mol, at least about 200 kJ/mol, at least about 250 kJ/mol, at least about 300 kJ/mol, at least about 350 kJ/mol, at least about 400 kJ/mol, at least about 450 kJ/mol, or at least about 500 kJ/mol.

[00269] Exemplary amino acid changes that can increase the stability of a CasX
variant protein relative to a reference CasX protein may include, but are not limited to, amino acid changes that increase the number of hydrogen bonds within the CasX variant protein, increase the number of disulfide bridges within the CasX variant protein, increase the number of salt bridges within the CasX variant protein, strengthen interactions between parts of the CasX
variant protein, increase the buried hydrophobic surface area of the CasX variant protein, or any combinations thereof.
k. Protein Yield

[00270] In some embodiments, the disclosure provides a CasX variant protein having improved yield during expression and purification relative to a reference CasX protein.
In some embodiments, the yield of CasX variant proteins purified from bacterial or eukaryotic host cells is improved relative to a reference CasX protein. In some embodiments, the bacterial host cells are Escherichia coil cells. In some embodiments, the eukaryotic cells are yeast, plant (e.g.
tobacco), insect (e.g. Spodoptera frugiperda sf9 cells), mouse, rat, hamster, guinea pig, non-human primate, or human cells. In some embodiments, the eukaryotic host cells are mammalian cells, including, but not limited to HEK293 cells, HEK293T cells, HEK293-F
cells, Lenti-X
293T cells, BHK cells, HepG2 cells, Saos-2 cells, HuH7 cells, A549 cells, NSO
cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, hybridoma cells, VERO cells, NIH3T3 cells, COS, WI38 cells, MRCS cells, HeLa, HT1080 cells, or CHO
cells.

[00271] In some embodiments, improved yield of the CasX variant protein is achieved through codon optimization. Cells use 64 different codons, 61 of which encode the 20 standard amino acids, while another 3 function as stop codons. In some cases, a single amino acid is encoded by more than one codon. Different organisms exhibit bias towards use of different codons for the same naturally occurring amino acid. Therefore, the choice of codons in a protein, and matching codon choice to the organism in which the protein will be expressed, can, in some cases, significantly affect protein translation and therefore protein expression levels. In some embodiments, the CasX variant protein is encoded by a nucleic acid that has been codon optimized. In some embodiments, the nucleic acid encoding the CasX variant protein has been codon optimized for expression in a bacterial cell, a yeast cell, an insect cell, a plant cell, or a mammalian cell. In some embodiments, the mammal cell is a mouse, a rat, a hamster, a guinea pig, a monkey, or a human. In some embodiments, the CasX variant protein is encoded by a nucleic acid that has been codon optimized for expression in a human cell. In some embodiments, the CasX variant protein is encoded by a nucleic acid from which nucleotide sequences that reduce translation rates in prokaryotes and eukaryotes have been removed. For example, runs of greater than three thymine residues in a row can reduce translation rates in certain organisms or internal polyadenylation signals can reduce translation.

[00272] In some embodiments, improvements in solubility and stability, as described herein, result in improved yield of the CasX variant protein relative to a reference CasX protein.

[00273] Improved protein yield during expression and purification can be evaluated by methods known in the art. For example, the amount of CasX variant protein can be determined by running the protein on an SDS-page gel, and comparing the CasX variant protein to a control whose amount or concentration is known in advance to determine an absolute level of protein.
Alternatively, or in addition, a purified CasX variant protein can be run on an SDS-page gel next to a reference CasX protein undergoing the same purification process to determine relative improvements in CasX variant protein yield. Alternatively, or in addition, levels of protein can be measured using immunohistochemical methods such as Western blot or ELISA
with an antibody to CasX, or by HPLC. For proteins in solution, concentration can be determined by measuring of the protein's intrinsic UV absorbance, or by methods which use protein-dependent color changes such as the Lowry assay, the Smith copper/bicinchoninic assay or the Bradford dye assay. Such methods can be used to calculate the total protein (such as, for example, total soluble protein) yield obtained by expression under certain conditions. This can be compared, for example, to the protein yield of a reference CasX protein under similar expression conditions.
1. Protein Solubility

[00274] In some embodiments, a CasX variant protein has improved solubility relative to a reference CasX protein. In some embodiments, a CasX variant protein has improved solubility of the CasX:gNA ribonucleoprotein complex variant relative to a ribonucleoprotein complex comprising a reference CasX protein.

[00275] In some embodiments, an improvement in protein solubility leads to higher yield of protein from protein purification techniques such as purification from E.
coil. Improved solubility of CasX variant proteins may, in some embodiments, enable more efficient activity in cells, as a more soluble protein may be less likely to aggregate in cells.
Protein aggregates can in certain embodiments be toxic or burdensome on cells, and, without wishing to be bound by any theory, increased solubility of a CasX variant protein may ameliorate this result of protein aggregation. Further, improved solubility of CasX variant proteins may allow for enhanced formulations permitting the delivery of a higher effective dose of functional protein, for example in a desired gene editing application. In some embodiments, improved solubility of a CasX
variant protein relative to a reference CasX protein results in improved yield of the CasX variant protein during purification of at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 250, at least about 500, or at least about 1000-fold greater yield. In some embodiments, improved solubility of a CasX variant protein relative to a reference CasX protein improves activity of the CasX variant protein in cells by at least about 1.1, at least about 1.2, at least about 1.3, at least about 1.4, at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, at least about 2, at least about 2.1, at least about 2.2, at least about 2.3, at least about 2.4, at least about 2.5, at least about 2.6, at least about 2.7, at least about 2.8, at least about 2.9, at least about 3, at least about 3.5, at least about 4, at least about 4.5, at least about 5, at least about 5.5, at least about 6, at least about 6.5, at least about 7.0, at least about 7.5, at least about 8, at least about 8.5, at least about 9, at least about 9.5, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15-fold, or at least about 20-fold greater activity.

[00276] Methods of measuring CasX protein solubility, and improvements thereof in CasX
variant proteins, will be readily apparent to the person of ordinary skill in the art. For example, CasX variant protein solubility can in some embodiments be measured by taking densitometry readings on a gel of the soluble fraction of lysed E.coli. Alternatively, or addition, improvements in CasX variant protein solubility can be measured by measuring the maintenance of soluble protein product through the course of a full protein purification, including the methods of the Examples. For example, soluble protein product can be measured at one or more steps of gel affinity purification, tag cleavage, cation exchange purification, running the protein on a size exclusion chromatography (SEC) column. In some embodiments, the densitometry of every band of protein on a gel is read after each step in the purification process.
CasX variant proteins with improved solubility may, in some embodiments, maintain a higher concentration at one or more steps in the protein purification process when compared to the reference CasX protein, while an insoluble protein variant may be lost at one or more steps due to buffer exchanges, filtration steps, interactions with a purification column, and the like.

[00277] In some embodiments, improving the solubility of CasX variant proteins results in a higher yield in terms of mg/L of protein during protein purification when compared to a reference CasX protein.

[00278] In some embodiments, improving the solubility of CasX variant proteins enables a greater amount of editing events compared to a less soluble protein when assessed in editing assays such as the EGFP disruption assays described herein.
m. Affinity for the gNA

[00279] In some embodiments, a CasX variant protein has improved affinity for the gNA
relative to a reference CasX protein, leading to the formation of the ribonucleoprotein complex.
Increased affinity of the CasX variant protein for the gNA may, for example, result in a lower Kd for the generation of a RNP complex, which can, in some cases, result in a more stable ribonucleoprotein complex formation. In some embodiments, the Kd of a CasX
variant protein for a gNA is increased relative to a reference CasX protein by a factor of at least about 1.1, at least about 1.2, at least about 1.3, at least about 1.4, at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100. In some embodiments, the CasX variant has about 1.1 to about 10-fold increased binding affinity to the gNA compared to the reference CasX
protein of SEQ ID NO: 2.

[00280] In some embodiments, increased affinity of the CasX variant protein for the gNA
results in increased stability of the ribonucleoprotein complex when delivered to mammalian cells, including in vivo delivery to a subject. This increased stability can affect the function and utility of the complex in the cells of a subject, as well as result in improved pharmacokinetic properties in blood, when delivered to a subject. In some embodiments, increased affinity of the CasX variant protein, and the resulting increased stability of the ribonucleoprotein complex, allows for a lower dose of the CasX variant protein to be delivered to the subject or cells while still having the desired activity; for example in vivo or in vitro gene editing. The increased ability to form RNP and keep them in stable form can be assessed using assays such as the in vitro cleavage assays described herein. In some embodiments, the CasX variants of the disclosure are able to achieve a Kcleave rate when complexed as an RNP that is at last 2-fold, at least 5-fold, or at least 10-fold higher compared to RNP of reference CasX.

[00281] In some embodiments, a higher affinity (tighter binding) of a CasX
variant protein to a gNA allows for a greater amount of editing events when both the CasX variant protein and the gNA remain in an RNP complex. Increased editing events can be assessed using editing assays such as the EGFP disruption and in vitro cleavage assays described herein.

[00282] Without wishing to be bound by theory, in some embodiments amino acid changes in the helical I domain can increase the binding affinity of the CasX variant protein with the gNA
targeting sequence, while changes in the helical II domain can increase the binding affinity of the CasX variant protein with the gNA scaffold stem loop, and changes in the oligonucleotide binding domain (OBD) increase the binding affinity of the CasX variant protein with the gNA
triplex.

[00283] Methods of measuring CasX protein binding affinity for a gNA include in vitro methods using purified CasX protein and gNA. The binding affinity for reference CasX and variant proteins can be measured by fluorescence polarization if the gNA or CasX protein is tagged with a fluorophore. Alternatively, or in addition, binding affinity can be measured by biolayer interferometry, electrophoretic mobility shift assays (EMSAs), or filter binding.
Additional standard techniques to quantify absolute affinities of RNA binding proteins such as the reference CasX and variant proteins of the disclosure for specific gNAs such as reference gNAs and variants thereof include, but are not limited to, isothermal calorimetry (ITC), and surface plasmon resonance (SPR), as well as the methods of the Examples.
n. Affinity for Target Nucleic Acid

[00284] In some embodiments, a CasX variant protein has improved binding affinity for a target nucleic acid relative to the affinity of a reference CasX protein for a target nucleic acid.
CasX variants with higher affinity for their target nucleic acid may, in some embodiments, cleave the target nucleic acid sequence more rapidly than a reference CasX
protein that does not have increased affinity for the target nucleic acid.

[00285] In some embodiments, the improved affinity for the target nucleic acid comprises improved affinity for the target sequence or protospacer sequence of the target nucleic acid, improved affinity for the PAM sequence, an improved ability to search DNA for the target sequence, or any combinations thereof. Without wishing to be bound by theory, it is thought that CRISPR/Cas system proteins such as CasX may find their target sequences by one-dimension diffusion along a DNA molecule. The process is thought to include (1) binding of the ribonucleoprotein to the DNA molecule followed by (2) stalling at the target sequence, either of which may be, in some embodiments, affected by improved affinity of CasX
proteins for a target nucleic acid sequence, thereby improving function of the CasX variant protein compared to a reference CasX protein.

[00286] In some embodiments, a CasX variant protein with improved target nucleic acid affinity has increased overall affinity for DNA. In some embodiments, a CasX
variant protein with improved target nucleic acid affinity has increased affinity for or the ability to utilize specific PAM sequences other than the canonical TTC PAM recognized by the reference CasX
protein of SEQ ID NO: 2, including PAM sequences selected from the group consisting of TTC, ATC, GTC, and CTC, thereby increasing the amount of target DNA that can be edited compared to wild-type CasX nucleases. Without wishing to be bound by theory, it is possible that these protein variants may interact more strongly with DNA overall and may have an increased ability to access and edit sequences within the target DNA due to the ability to utilize additional PAM
sequences beyond those of wild-type reference CasX, thereby allowing for a more efficient search process of the CasX protein for the target sequence. A higher overall affinity for DNA
also, in some embodiments, can increase the frequency at which a CasX protein can effectively start and finish a binding and unwinding step, thereby facilitating target strand invasion and R-loop formation, and ultimately the cleavage of a target nucleic acid sequence.

[00287] Without wishing to be bound by theory, it is possible that amino acid changes in the NTSBD that increase the efficiency of unwinding, or capture, of a non-target DNA strand in the unwound state, can increase the affinity of CasX variant proteins for target DNA. Alternatively, or in addition, amino acid changes in the NTSBD that increase the ability of the NTSBD to stabilize DNA during unwinding can increase the affinity of CasX variant proteins for target DNA. Alternatively, or in addition, amino acid changes in the OBD may increase the affinity of CasX variant protein binding to the protospacer adjacent motif (PAM), thereby increasing affinity of the CasX variant protein for target nucleic acid. Alternatively, or in addition, amino acid changes in the Helical I and/or II, RuvC and TSL domains that increase the affinity of the CasX variant protein for the target nucleic acid strand can increase the affinity of the CasX
variant protein for target nucleic acid.

[00288] In some embodiments, binding affinity of a CasX variant protein of the disclosure for a target nucleic acid molecule is increased relative to a reference CasX protein by a factor of at least about 1.1, at least about 1.2, at least about 1.3, at least about 1.4, at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100. In some embodiments, the CasX variant protein has about 1.1 to about 100-fold increased binding affinity to the target nucleic acid compared to the reference protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID
NO: 3.

[00289] In some embodiments, a CasX variant protein has improved binding affinity for the non-target strand of the target nucleic acid. As used herein, the term "non-target strand" refers to the strand of the DNA target nucleic acid sequence that does not form Watson and Crick base pairs with the targeting sequence in the gNA, and is complementary to the target DNA strand.
In some embodiments, the CasX variant protein has about 1.1 to about 100-fold increased binding affinity to the non-target stand of the target nucleic acid compared to the reference protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[00290] Methods of measuring CasX protein (such as reference or variant) affinity for a target and/or non-target nucleic acid molecule may include electrophoretic mobility shift assays (EMSAs), filter binding, isothermal calorimetry (ITC), and surface plasmon resonance (SPR), fluorescence polarization and biolayer interferometry (BLI). Further methods of measuring CasX protein affinity for a target include in vitro biochemical assays that measure DNA
cleavage events over time.
o. Improved Specificity for a Target Site

[00291] In some embodiments, a CasX variant protein has improved specificity for a target nucleic acid sequence relative to a reference CasX protein. As used herein, "specificity,"
sometimes referred to as "target specificity," refers to the degree to which a CRISPR/Cas system ribonucleoprotein complex cleaves off-target sequences that are similar, but not identical to the target nucleic acid sequence; e.g., a CasX variant RNP with a higher degree of specificity would exhibit reduced off-target cleavage of sequences relative to a reference CasX
protein. The specificity, and the reduction of potentially deleterious off-target effects, of CRISPR/Cas system proteins can be vitally important in order to achieve an acceptable therapeutic index for use in mammalian subjects.

[00292] In some embodiments, a CasX variant protein has improved specificity for a target site within the target sequence that is complementary to the targeting sequence of the gNA. Without wishing to be bound by theory, it is possible that amino acid changes in the helical I and II
domains that increase the specificity of the CasX variant protein for the target nucleic acid strand can increase the specificity of the CasX variant protein for the target nucleic acid overall. In some embodiments, amino acid changes that increase specificity of CasX variant proteins for target nucleic acid may also result in decreased affinity of CasX variant proteins for DNA.

[00293] Methods of testing CasX protein (such as variant or reference) target specificity may include guide and Circularization for In vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq), or similar methods. In brief, in CIRCLE-seq techniques, genomic DNA is sheared and circularized by ligation of stem-loop adapters, which are nicked in the stem-loop regions to expose 4 nucleotide palindromic overhangs. This is followed by intramolecular ligation and degradation of remaining linear DNA. Circular DNA molecules containing a CasX
cleavage site are subsequently linearized with CasX, and adapter adapters are ligated to the exposed ends followed by high-throughput sequencing to generate paired end reads that contain information about the off-target site. Additional assays that can be used to detect off-target events, and therefore CasX protein specificity include assays used to detect and quantify indels (insertions and deletions) formed at those selected off-target sites such as mismatch-detection nuclease assays and next generation sequencing (NGS). Exemplary mismatch-detection assays include nuclease assays, in which genomic DNA from cells treated with CasX and sgNA is PCR
amplified, denatured and rehybridized to form hetero-duplex DNA, containing one wild type strand and one strand with an indel. Mismatches are recognized and cleaved by mismatch detection nucleases, such as Surveyor nuclease or T7 endonuclease I.
p. Protospacer and PAM Sequences

[00294] Herein, the protospacer is defined as the DNA sequence complementary to the targeting sequence of the guide RNA and the DNA complementary to that sequence, referred to as the target strand and non-target strand, respectively. As used herein, the PAM is a nucleotide sequence proximal to the protospacer that, in conjunction with the targeting sequence of the gNA, helps the orientation and positioning of the CasX for the potential cleavage of the protospacer strand(s).

[00295] PAM sequences may be degenerate, and specific RNP constructs may have different preferred and tolerated PAM sequences that support different efficiencies of cleavage. Following convention, unless stated otherwise, the disclosure refers to both the PAM and the protospacer sequence and their directionality according to the orientation of the non-target strand. This does not imply that the PAM sequence of the non-target strand, rather than the target strand, is determinative of cleavage or mechanistically involved in target recognition.
For example, when reference is to a TTC PAM, it may in fact be the complementary GAA sequence that is required for target cleavage, or it may be some combination of nucleotides from both strands. In the case of the CasX proteins disclosed herein, the PAM is located 5' of the protospacer with a single nucleotide separating the PAM from the first nucleotide of the protospacer.
Thus, in the case of reference CasX, a TTC PAM should be understood to mean a sequence following the formula 5' -...NNTTCN(protospacer) ...3' (SEQ ID NO: 3296) where 'N' is any DNA
nucleotide and '(protospacer)' is a DNA sequence having identity with the targeting sequence of the guide RNA. In the case of a CasX variant with expanded PAM recognition, a TTC, CTC, GTC, or ATC PAM should be understood to mean a sequence following the formulae: 5'-...NNTTCN(protospacer) ...3' (SEQ ID NO: 3296); 5'-...NNCTCN(protospacer) ...3' (SEQ ID NO: 3297); 5'-...NNGTCN(protospacer) ...3' (SEQ ID NO: 3298); or 5'-...NNATCN(protospacer) ...3' (SEQ ID NO: 3299). Alternatively, a TC PAM
should be understood to mean a sequence following the formula 5'-...NNNTCN(protospacer) ...3' (SEQ ID NO: 3300).

[00296] In some embodiments, a CasX variant has improved editing of a PAM
sequence exhibits greater editing efficiency and/or binding of a target sequence in the target DNA when any one of the PAM sequences TTC, ATC, GTC, or CTC is located 1 nucleotide 5' to the non-target strand of the protospacer having identity with the targeting sequence of the gNA in a cellular assay system compared to the editing efficiency and/or binding of an RNP comprising a reference CasX protein in a comparable assay system. In some embodiments, the PAM sequence is TTC. In some embodiments, the PAM sequence is ATC. In some embodiments, the PAM
sequence is CTC. In some embodiments, the PAM sequence is GTC.
q. Unwinding of DNA

[00297] In some embodiments, a CasX variant protein has improved ability to unwind DNA
relative to a reference CasX protein. Poor dsDNA unwinding has been shown previously to impair or prevent the ability of CRISPR/Cas system proteins AnaCas9 or Cas14s to cleave DNA. Therefore, without wishing to be bound by any theory, it is likely that increased DNA
cleavage activity by some CasX variant proteins of the disclosure is due, at least in part, to an increased ability to find and unwind the dsDNA at a target site. Methods of measuring the ability of CasX proteins (such as variant or reference) to unwind DNA include, but are not limited to, in vitro assays that observe increased on rates of dsDNA targets in fluorescence polarization or biolayer interferometry.

[00298] Without wishing to be bound by theory, it is thought that amino acid changes in the NTSB domain may produce CasX variant proteins with increased DNA unwinding characteristics. Alternatively, or in addition, amino acid changes in the OBD
or the helical domain regions that interact with the PAM may also produce CasX variant proteins with increased DNA unwinding characteristics.
r. Catalytic Activity

[00299] The ribonucleoprotein complex of the CasX:gNA systems disclosed herein comprise a reference CasX protein or CasX variant complexed with a gNA that binds to a target nucleic acid and, in some cases, cleaves the target nucleic acid. In some embodiments, a CasX variant protein has improved catalytic activity relative to a reference CasX protein.
Without wishing to be bound by theory, it is thought that in some cases cleavage of the target strand can be a limiting factor for Cas12-like molecules in creating a dsDNA break. In some embodiments, CasX variant proteins improve bending of the target strand of DNA and cleavage of this strand, resulting in an improvement in the overall efficiency of dsDNA cleavage by the CasX
ribonucleoprotein complex.

[00300] In some embodiments, a CasX variant protein has increased nuclease activity compared to a reference CasX protein. Variants with increased nuclease activity can be generated, for example, through amino acid changes in the RuvC nuclease domain. In some embodiments, amino acid substitutions in amino acid residues 708-804 of the RuvC domain can result in increased editing efficiency, as seen in FIG. 10. In some embodiments, the CasX variant comprises a nuclease domain having nickase activity. In the foregoing embodiment, the CasX
nickase of a gene editing pair generates a single-stranded break within 10-18 nucleotides 3' of a PAM site in the non-target strand. In other embodiments, the CasX variant comprises a nuclease domain having double-stranded cleavage activity. In the foregoing, the CasX of the gene editing pair generates a double-stranded break within 18-26 nucleotides 5' of a PAM
site on the target strand and 10-18 nucleotides 3' on the non-target strand. Nuclease activity can be assayed by a variety of methods, including those of the Examples. In some embodiments, a CasX variant has a Kcleave constant that is at least 2-fold, or at least 3-fold, or at least 4-fold, or at least 5-fold, or at least 6-fold, or at least 7-fold, or at least 8-fold, or at least 9-fold, or at least 10-fold greater compared to a reference or wild-type CasX.

[00301] In some embodiments, a CasX variant protein has increased target strand loading for double strand cleavage. Variants with increased target strand loading activity can be generated, for example, through amino acid changes in the TLS domain. Without wishing to be bound by theory, amino acid changes in the TSL domain may result in CasX variant proteins with improved catalytic activity. Alternatively, or in addition, amino acid changes around the binding channel for the RNA:DNA duplex may also improve catalytic activity of the CasX
variant protein.

[00302] In some embodiments, a CasX variant protein has increased collateral cleavage activity compared to a reference CasX protein. As used herein, "collateral cleavage activity" refers to additional, non-targeted cleavage of nucleic acids following recognition and cleavage of a target nucleic acid. In some embodiments, a CasX variant protein has decreased collateral cleavage activity compared to a reference CasX protein.

[00303] In some embodiments, for example those embodiments encompassing applications where cleavage of the target nucleic acid is not a desired outcome, improving the catalytic activity of a CasX variant protein comprises altering, reducing, or abolishing the catalytic activity of the CasX variant protein. In some embodiments, a ribonucleoprotein complex comprising a dCasX variant protein binds to a target nucleic acid and does not cleave the target nucleic acid.

[00304] In some embodiments, the CasX ribonucleoprotein complex comprising a CasX variant protein binds a target DNA but generates a single stranded nick in the target DNA. In some embodiments, particularly those embodiments wherein the CasX protein is a nickase, a CasX
variant protein has decreased target strand loading for single strand nicking.
Variants with decreased target strand loading may be generated, for example, through amino acid changes in the TSL domain.

[00305] Exemplary methods for characterizing the catalytic activity of CasX
proteins may include, but are not limited to, in vitro cleavage assays, including those of the Examples, below.
In some embodiments, electrophoresis of DNA products on agarose gels can interrogate the kinetics of strand cleavage.
s. Affinity for Target RNA

[00306] In some embodiments, a ribonucleoprotein complex comprising a reference CasX
protein or variant thereof binds to a target RNA and cleaves the target nucleic acid. In some embodiments, variants of a reference CasX protein increase the specificity of the CasX variant protein for a target RNA, and increase the activity of the CasX variant protein with respect to a target RNA when compared to the reference CasX protein. For example, CasX
variant proteins can display increased binding affinity for target RNAs, or increased cleavage of target RNAs, when compared to reference CasX proteins. In some embodiments, a ribonucleoprotein complex comprising a CasX variant protein binds to a target RNA and/or cleaves the target RNA. In some embodiments, a CasX variant has at least about two-fold to about 10-fold increased binding affinity to the target nucleic acid compared to the reference protein of SEQ
ID NO: 1, SEQ ID
NO: 2, or SEQ ID NO: 3.

t. CasX Fusion Proteins

[00307] In some embodiments, the disclosure provides CasX proteins comprising a heterologous protein fused to the CasX. In some cases, the CasX is a reference CasX protein. In other cases, the CasX is a CasX variant of any of the embodiments described herein.

[00308] In some embodiments, the CasX variant protein is fused to one or more proteins or domains thereof that have a different activity of interest, resulting in a fusion protein. For example, in some embodiments, the CasX variant protein is fused to a protein (or domain thereof) that inhibits transcription, modifies a target nucleic acid, or modifies a polypeptide associated with a nucleic acid (e.g., histone modification).

[00309] In some embodiments, a CasX variant comprises any one of SEQ ID NOS:
247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4412-4415 fused to one or more proteins or domains thereof with an activity of interest. In some embodiments, a CasX
variant comprises any one of SEQ ID NOS: 247-337, 3498-3501, 3505-3520, 3540-3549 and 4412-4415 fused to one or more proteins or domains thereof with an activity of interest. In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 3498-3501, 3505-3520, and 3540-3549 fused to one or more proteins or domains thereof with an activity of interest.

[00310] In some embodiments, a heterologous polypeptide (or heterologous amino acid such as a cysteine residue or a non-natural amino acid) can be inserted at one or more positions within a CasX protein to generate a CasX fusion protein. In other embodiments, a cysteine residue can be inserted at one or more positions within a CasX protein followed by conjugation of a heterologous polypeptide described below. In some alternative embodiments, a heterologous polypeptide or heterologous amino acid can be added at the N- or C-terminus of the reference or CasX variant protein. In other embodiments, a heterologous polypeptide or heterologous amino acid can be inserted internally within the sequence of the CasX protein.

[00311] In some embodiments, the reference CasX or variant fusion protein retains RNA-guided sequence specific target nucleic acid binding and cleavage activity. In some cases, the reference CasX or variant fusion protein has (retains) 50% or more of the activity (e.g., cleavage and/or binding activity) of the corresponding reference CasX or variant protein that does not have the insertion of the heterologous protein. In some cases, the reference CasX or variant fusion protein retains at least about 60%, or at least about 70%, at least about 80%, or at least about 90%, or at least about 92%, or at least about 95%, or at least about 98%, or about 100% of the activity (e.g., cleavage and/or binding activity) of the corresponding CasX protein that does not have the insertion of the heterologous protein.

[00312] In some cases, the reference CasX or CasX variant fusion protein retains (has) target nucleic acid binding activity relative to the activity of the CasX protein without the inserted heterologous amino acid or heterologous polypeptide. In some cases, the reference CasX or CasX variant fusion protein retains at least about 60%, or at least about 70%, at least about 80%, or at least about 90%, or at least about 92%, or at least about 95%, or at least about 98%, or about 100% of the binding activity of the corresponding CasX protein that does not have the insertion of the heterologous protein.

[00313] In some cases, the reference CasX or CasX variant fusion protein retains (has) target nucleic acid binding and/or cleavage activity relative to the activity of the parent CasX protein without the inserted heterologous amino acid or heterologous polypeptide. For example, in some cases, the reference CasX or CasX variant fusion protein has (retains) 50% or more of the binding and/or cleavage activity of the corresponding parent CasX protein (the CasX protein that does not have the insertion). For example, in some cases, the reference CasX
or CasX variant fusion protein has (retains) 60% or more (70% or more, 80% or more, 90% or more, 92% or more, 95% or more, 98% or more, or 100%) of the binding and/or cleavage activity of the corresponding CasX parent protein (the CasX protein that does not have the insertion). Methods of measuring cleaving and/or binding activity of a CasX protein and/or a CasX
fusion protein will be known to one of ordinary skill in the art, and any convenient method can be used.

[00314] A variety of heterologous polypeptides are suitable for inclusion in a reference CasX or CasX variant fusion protein of the disclosure. In some cases, the fusion partner can modulate transcription (e.g., inhibit transcription, increase transcription) of a target DNA. For example, in some cases the fusion partner is a protein (or a domain from a protein) that inhibits transcription (e.g., a transcriptional repressor, a protein that functions via recruitment of transcription inhibitor proteins, modification of target DNA such as methylation, recruitment of a DNA
modifier, modulation of histones associated with target DNA, recruitment of a histone modifier such as those that modify acetylation and/or methylation of histones, and the like).
In some cases the fusion partner is a protein (or a domain from a protein) that increases transcription (e.g., a transcription activator, a protein that acts via recruitment of transcription activator proteins, modification of target DNA such as demethylation, recruitment of a DNA
modifier, modulation of histones associated with target DNA, recruitment of a hi stone modifier such as those that modify acetylation and/or methylation of histones, and the like).

[00315] In some cases, a fusion partner has enzymatic activity that modifies a target nucleic acid; e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity.

[00316] In some cases, a fusion partner has enzymatic activity that modifies a polypeptide (e.g., a histone) associated with a target nucleic acid; e.g., methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity. In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, and 4412-4415 and a polypeptide with methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity. In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 247-337, 3498-3501, 3505-3520, 3540-3549 and and a polypeptide with methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity. In some embodiments, a CasX variant comprises any one of SEQ ID
NOS: 3498-3501, 3505-3520, and 3540-3549 and a polypeptide with methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity.

[00317] Examples of proteins (or fragments thereof) that can be used as a suitable fusion partner to a reference CasX or CasX variant to increase transcription include but are not limited to: transcriptional activators such as VP16, VP64, VP48, VP160, p65 subdomain (e.g., from NFkB), and activation domain of EDLL and/or transcription activator-like (TAL) activation domain (e.g., for activity in plants); histone lysine methyltransferases such as SET domain containing 1A, histone lysine methyltransferase (SET1A), SET domain containing 1B, histone lysine methyltransferase (SET1B), lysine methyltransferase 2A (MLL1) to 5, ASCL1 (ASH1) achaete-scute family bHLH transcription factor 1 (ASH1), SET and MYND domain containing 2provided (SMYD2), nuclear receptor binding SET domain protein 1 (NSD1), and the like;
histone lysine demethylases such as lysine demethylase 3A (JHDM2a)/ Lysine-specific demethylase 3B (JHDM2b), lysine demethylase 6A (UTX), lysine demethylase 6B
(JMJD3), and the like; histone acetyltransferases such as lysine acetyltransferase 2A
(GCN5), lysine acetyltransferase 2B (PCAF), CREB binding protein (CBP), El A binding protein p30 (p300), TATA-box binding protein associated factor 1 (TAF1), lysine acetyltransferase 5 (TIP60/PLIP), lysine acetyltransferase 6A (MOZ/MYST3), lysine acetyltransferase 6B
(MORF/MYST4), SRC
proto-oncogene, non-receptor tyrosine kinase (SRC1), nuclear receptor coactivator 3 (ACTR), MYB binding protein la (P160), clock circadian regulator (CLOCK), and the like; and DNA
demethylases such as Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), tet methylcytosine dioxygenase 1 (TETI), demeter (DME), demeter-like 1 (DML1), demeter-like 2 (DML2), protein ROS1 (ROS1), and the like.

[00318] Examples of proteins (or fragments thereof) that can be used as a suitable fusion partner with a reference CasX or CasX variant to decrease transcription include but are not limited to: transcriptional repressors such as the Kruppel associated box (KRAB or SKD);
KOX1 repression domain; the Mad mSIN3 interaction domain (SID); the ERF
repressor domain (ERD), the SRDX repression domain (e.g., for repression in plants), and the like; histone lysine methyltransferases such as PR/SET domain containing protein (Pr-SET)7/8, lysine methyltransferase 5B (SUV4- 20H1), PR/SET domain 2 (RIZ1), and the like;
histone lysine demethylases such as lysine demethylase 4A (JMJD2A/JHDM3A), lysine demethylase (JMJD2B), lysine demethylase 4C (JMJD2C/GASC1), lysine demethylase 4D
(JMJD2D), lysine demethylase 5A (JARID1A/RBP2), lysine demethylase 5B (JARID1B/PLU-1), lysine demethylase 5C (JARID 1C/SMCX), lysine demethylase 5D (JARID1D/SMCY), and the like;
histone lysine deacetylases such as histone deacetylase 1 (HDAC1), HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, sirtuin 1 (SIRT1), SIRT2, HDAC11, and the like;
DNA
methylases such as HhaI DNA m5c-methyltransferase (M.HhaI), DNA
methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), methyltransferase 1 (MET1), S-adenosyl-L-methionine-dependent methyltransferases superfamily protein (DRM3) (plants), DNA cytosine methyltransferase MET2a (ZMET2), chromomethylase 1 (CMT1), chromomethylase 2 (CMT2) (plants), and the like; and periphery recruitment elements such as Lamin A, Lamin B, and the like.

[00319] In some cases, the fusion partner to a reference CasX or CasX variant has enzymatic activity that modifies the target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA). Examples of enzymatic activity that can be provided by the fusion partner include but are not limited to:
nuclease activity such as that provided by a restriction enzyme (e.g., FokI
nuclease), methyltransferase activity such as that provided by a methyltransferase (e.g., Hhal DNA m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA
methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants), and the like); demethylase activity such as that provided by a demethylase (e.g., Ten-Eleven Translocation (TET) dioxygenase 1 (TET 1 CD), TETI, DME, DML1, DML2, ROS1, and the like), DNA repair activity, DNA damage activity, deamination activity such as that provided by a deaminase (e.g., a cytosine deaminase enzyme, e.g., an APOBEC protein such as rat apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1 {APOBEC1}), dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity such as that provided by an integrase and/or resolvase (e.g., Gin invertase such as the hyperactive mutant of the Gin invertase, GinH106Y; human immunodeficiency virus type 1 integrase (IN); Tn3 resolvase; and the like), transposase activity, recombinase activity such as that provided by a recombinase (e.g., catalytic domain of Gin recombinase), polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity).

[00320] In some cases, a reference CasX or CasX variant protein of the present disclosure is fused to a polypeptide selected from: a domain for increasing transcription (e.g., a VP16 domain, a VP64 domain), a domain for decreasing transcription (e.g., a KRAB domain, e.g., from the Koxl protein), a core catalytic domain of a hi stone acetyltransferase (e.g., histone acetyltransferase p300), a protein/domain that provides a detectable signal (e.g., a fluorescent protein such as GFP), a nuclease domain (e.g., a Fokl nuclease), and a base editor (discussed further below).

[00321] In some embodiments, a CasX variant comprises any one of SEQ ID NOS:
247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4412-4415 fused to a polypeptide selected from the group consisting of a domain for decreasing transcription, a domain with enzymatic activity, a core catalytic domain of a histone acetyltransferase, a protein/domain that provides a detectable signal, a nuclease domain, and a base editor. In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 247-337, 3498-3501, 3505-3520, 3540-3549 and fused to a polypeptide selected from the group consisting of a domain for decreasing transcription, a domain with enzymatic activity, a core catalytic domain of a hi stone acetyltransferase, a protein/domain that provides a detectable signal, a nuclease domain, and a base editor. In some embodiments, a CasX variant comprises any one of SEQ ID
NOS: 3498-3501, 3505-3520, and 3540-3549 fused to a polypeptide selected from the group consisting of a domain for decreasing transcription, a domain with enzymatic activity, a core catalytic domain of a histone acetyltransferase, a protein/domain that provides a detectable signal, a nuclease domain, and a base editor.

[00322] In some cases, a reference CasX protein or CasX variant of the present disclosure is fused to a base editor. Base editors include those that can alter a guanine, adenine, cytosine, thymine, or uracil base on a nucleoside or nucleotide. Base editors include, but are not limited to an adenosine deaminase, cytosine deaminase (e.g. APOBEC1), and guanine oxidase.
Accordingly, any of the reference CasX or CasX variants provided herein may comprise (i.e., are fused to) a base editor; for example a reference CasX or CasX variant of the disclosure may be fused to an adenosine deaminase, a cytosine deaminase, or a guanine oxidase.
In exemplary embodiments, a CasX variant of the disclosure comprising any one of SEQ ID
NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4412-4415 is fused to an adenosine deaminase, cytosine deaminase, or a guanine oxidase.

[00323] In some cases, the fusion partner to a reference CasX or CasX variant has enzymatic activity that modifies a protein associated with the target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA) (e.g., a histone, an RNA binding protein, a DNA binding protein, and the like).
Examples of enzymatic activity (that modifies a protein associated with a target nucleic acid) that can be provided by the fusion partner with a reference CasX or CasX
variant include but are not limited to: methyltransferase activity such as that provided by a histone methyltransferase (HMT) (e.g., suppressor of variegation 3-9 homolog 1 (SUV39H1, also known as KMT1A), euchromatic histone lysine methyltransferase 2 (G9A, also known as KMT1C and EHMT2), SUV39H2, ESET/SETDB 1, and the like, SET1A, SET1B, MLL1 to 5, ASH1, SMYD2, NSD1, DOTI like histone lysine methyltransferase (DOT1L), Pr-SET7/8, lysine methyltransferase 5B
(SUV4-20H1), enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), PR/SET
domain 2 (RIZ1), demethylase activity such as that provided by a histone demethylase (e.g., Lysine Demethylase 1A (KDM1A also known as LSD1), JHDM2a/b, JMJD2A/JHDM3A, JMJD2B, JMJD2C/GASC1, JMJD2D, JARID1A/RBP2, JARID1B/PLU-1, JARID1C/SMCX, JARID1D/SMCY, UTX, JMJD3, and the like), acetyltransferase activity such as that provided by a histone acetylase transferase (e.g., catalytic core/fragment of the human acetyltransferase p300, GCN5, PCAF, CBP, TAF1, TIP60/PLIP, MOZ/MYST3, MORF/MYST4, HB01/MYST2, HMOF/MYST1, SRC1, ACTR, P160, CLOCK, and the like), deacetylase activity such as that provided by a histone deacetylase (e.g., HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, and the like), kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, and demyristoylation activity.

[00324] Additional examples of suitable fusion partners to a reference CasX or CasX variant are (i) a dihydrofolate reductase (DHFR) destabilization domain (e.g., to generate a chemically controllable subject RNA-guided polypeptide), and (ii) a chloroplast transit peptide.

[00325] Suitable chloroplast transit peptides include, but are not limited to sequences having at least 80%, at least 90%, or at least 95% identity to or are identical to:
MASMISSSAVTTVSRASRGQSAAMAPFGGLKSMTGFPVRKVNTDITSITSNGGR
VKCMQVWPPIGKKKFETLSYLPPLTRDSRA (SEQ ID NO: 338);
MASMISSSAVTTVSRASRGQSAAMAPFGGLKSMTGFPVRKVNTDITSITSNGGRVKS
(SEQ ID NO: 339);
MASSMLSSATMVASPAQATMVAPFNGLKSSAAFPATRKANNDITSITSNGGRVNCMQV
WPPIEKKKFETLSYLPDLTDSGGRVNC (SEQ ID NO: 340);
MAQVSRICNGVQNPSLISNLSKSSQRKSPLSVSLKTQQHPRAYPISSSWGLKKSGMTLIG
SELRPLKVMSSVSTAC (SEQ ID NO: 341);
MAQVSRICNGVWNPSLISNLSKSSQRKSPLSVSLKTQQHPRAYPISSSWGLKKSGMTLIG
SELRPLKVMSSVSTAC (SEQ ID NO: 342);

MAQINNMAQGIQTLNPNSNFHKPQVPKSSSFLVFGSKKLKNSANSMLVLKKDSIFMQLF
CSFRISASVATAC (SEQ ID NO: 343);
MAALVTSQLATSGTVLSVTDRFRRPGFQGLRPRNPADAALGMRTVGASAAPKQSRKPH
RFDRRCLSMVV (SEQ ID NO: 344);
MAALTTSQLATSATGFGIADRSAPSSLLRHGFQGLKPRSPAGGDATSLSVTTSARATPKQ
QRSVQRGSRRFPSVVVC (SEQ ID NO: 345);
MASSVLSSAAVATRSNVAQANMVAPFTGLKSAASFPVSRKQNLDITSIASNGGRVQC
(SEQ ID NO: 346);
MESLAATSVFAPSRVAVPAARALVRAGTVVPTRRTSSTSGTSGVKCSAAVTPQASPVIS
RSAAAA (SEQ ID NO: 347); and MGAAATSMQSLKFSNRLVPPSRRLSPVPNNVTCNNLPKSAAPVRTVKCCASSWNSTING
AAATTNGASAASS (SEQ ID NO: 348). In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and and a chloroplast transit peptide. In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 247-337, 3498-3501, 3505-3520, 3540-3549 and 4412-4415 and a chloroplast transit peptide. In some embodiments, a CasX variant comprises any one of SEQ
ID NOS: 3498-3501, 3505-3520, and 3540-3549 and a chloroplast transit peptide.

[00326] In some cases, a reference CasX or CasX variant protein of the present disclosure can include an endosomal escape peptide. In some cases, an endosomal escape polypeptide comprises the amino acid sequence GLFXALLXLLXSLWXLLLXA (SEQ ID NO: 349), wherein each X is independently selected from lysine, histidine, and arginine.
In some cases, an endosomal escape polypeptide comprises the amino acid sequence GLFHALLHLLHSLWHLLLHA (SEQ ID NO: 350), or HEIHHHHEIHH (SEQ ID NO: 351). In some embodiments, a CasX variant comprises a sequence of any one of SEQ ID
NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4412-4415 and an endosomal escape polypeptide. In some embodiments, a CasX variant comprises a sequence of any one of SEQ ID
NOS: 247-337, 3498-3501, 3505-3520, 3540-3549 and 4412-4415 and an endosomal escape polypeptide. In some embodiments, a CasX variant comprises a sequence of any one of SEQ ID
NOS: 3498-3501, 3505-3520, and 3540-3549 and an endosomal escape polypeptide.

[00327] Non-limiting examples of suitable fusion partners for a reference CasX
or CasX variant for use when targeting ssRNA target nucleic acids include (but are not limited to): splicing factors (e.g., RS domains); protein translation components (e.g., translation initiation, elongation, and/or release factors; e.g., eukaryotic translation initiation factor 4 gamma {eIF4G}); RNA methylases; RNA editing enzymes (e.g., RNA deaminases, e.g., adenosine deaminase acting on RNA (ADAR), including A to I and/or C to U editing enzymes); helicases;
RNA-binding proteins; and the like. It is understood that a heterologous polypeptide can include the entire protein or in some cases can include a fragment of the protein (e.g., a functional domain). In some embodiments, a CasX variant comprises any one of SEQ ID NOS:
247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4412-4415 and a protein or domain selected from the group consisting of a splicing factor, a protein translation component, an RNA
methylase, an RNA editing enzyme, a helicase, and an RNA binding protein. In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 247-337, 3498-3501, 3505-3520, 3540-3549 and 4412-4415 and a protein or domain selected from the group consisting of a splicing factor, a protein translation component, an RNA methylase, an RNA
editing enzyme, a helicase, and an RNA binding protein. In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 3498-3501, 3505-3520, and 3540-3549 and a protein or domain selected from the group consisting of a splicing factor, a protein translation component, an RNA methylase, an RNA editing enzyme, a helicase, and an RNA binding protein..

[00328] A fusion partner for a reference CasX or CasX variant can be any domain capable of interacting with ssRNA (which, for the purposes of this disclosure, includes intramolecular and/or intermolecular secondary structures, e.g., double-stranded RNA duplexes such as hairpins, stem-loops, etc.), whether transiently or irreversibly, directly or indirectly, including but not limited to an effector domain selected from the group comprising;
endonucleases (for example RNase III, the CRR22 DYW domain, Dicer, and PIN (PilT N-terminus) domains from proteins such as SMG5 and SMG6); proteins and protein domains responsible for stimulating RNA cleavage (for example cleavage and polyadenylation specific factor {CPSF}, cleavage stimulation factor {CstF}, CFIm and CFIIm); exonucleases (for example chromatin-binding exonuclease XRN1 (XRN-1) or Exonuclease T); deadenylases (for example DNA 5'-adenosine monophosphate hydrolase {I-INT3}); proteins and protein domains responsible for nonsense mediated RNA decay (for example UPF1 RNA helicase and ATPase {UPF1}, UPF2, UPF3, UPF3b, RNP SI, RNA binding motif protein 8A {Y14}, DEK proto-oncogene {DEK}, RNA-processing protein REF2 {REF2}, and Serine-arginine repetitive matrix 1 {SRm160}); proteins and protein domains responsible for stabilizing RNA (for example poly(A) binding protein cytoplasmic 1 {PABP}); proteins and protein domains responsible for repressing translation (for example argonaute RISC catalytic component 2 {Ago2} and Ago4); proteins and protein domains responsible for stimulating translation (for example Staufen);
proteins and protein domains responsible for (e.g., capable of) modulating translation (e.g., translation factors such as initiation factors, elongation factors, release factors, etc., e.g., eIF4G);
proteins and protein domains responsible for polyadenylation of RNA (for example poly(A) polymerase (PAP1), PAP-associated domain-containing protein;Poly(A) RNA polymerase gld-2 {GLD-2}, and Star-PAP); proteins and protein domains responsible for polyuridinylation of RNA
(for example Terminal uridylyltransferase {CID1} and terminal uridylate transferase);
proteins and protein domains responsible for RNA localization (for example from insulin like growth factor 2 mRNA
binding protein 1 {IMP1}, Z-DNA binding protein 1 {ZBP1}, 5he2p, 5he3p, and Bicaudal-D);
proteins and protein domains responsible for nuclear retention of RNA (for example Rrp6);
proteins and protein domains responsible for nuclear export of RNA (for example nuclear RNA
export factor 1 {TAP}, nuclear RNA export factor 1 {NXF1}, THO Complex {THO}, TREX, REF, and Aly/REF export factor {Aly}); proteins and protein domains responsible for repression of RNA splicing (for example polypyrimidine tract binding protein 1 {PTB}, KH
RNA binding domain containing, signal transduction associated 1 5am681, and heterogeneous nuclear ribonucleoprotein Al {11nRNP Al}); proteins and protein domains responsible for stimulation of RNA splicing (for example serine/arginine-rich (SR) domains); proteins and protein domains responsible for reducing the efficiency of transcription (for example FUS RNA
binding protein {FUS (TLS)}); and proteins and protein domains responsible for stimulating transcription (for example cyclin dependent kinase 7 {CDK7} and HIV Tat). Alternatively, the effector domain may be selected from the group comprising endonucleases; proteins and protein domains capable of stimulating RNA cleavage; exonucleases; deadenylases; proteins and protein domains having nonsense mediated RNA decay activity; proteins and protein domains capable of stabilizing RNA; proteins and protein domains capable of repressing translation; proteins and protein domains capable of stimulating translation; proteins and protein domains capable of modulating translation (e.g., translation factors such as initiation factors, elongation factors, release factors, etc., e.g., elF4G); proteins and protein domains capable of polyadenylation of RNA; proteins and protein domains capable of polyuridinylation of RNA; proteins and protein domains having RNA localization activity; proteins and protein domains capable of nuclear retention of RNA;
proteins and protein domains having RNA nuclear export activity; proteins and protein domains capable of repression of RNA splicing; proteins and protein domains capable of stimulation of RNA splicing; proteins and protein domains capable of reducing the efficiency of transcription;
and proteins and protein domains capable of stimulating transcription. Another suitable heterologous polypeptide is a PUF RNA-binding domain, which is described in more detail in W02012068627, which is hereby incorporated by reference in its entirety.

[00329] Some suitable RNA splicing factors that can be used (in whole or as fragments thereof) as a fusion partner with a reference CasX or CasX variant have modular organization, with separate sequence-specific RNA binding modules and splicing effector domains.
For example, members of the serine/arginine-rich (SR) protein family contain N-terminal RNA
recognition motifs (RRMs) that bind to exonic splicing enhancers (ESEs) in pre-mRNAs and C-terminal RS
domains that promote exon inclusion. As another example, the hnRNP protein hnRNP Al binds to exonic splicing silencers (ESSs) through its RRM domains and inhibits exon inclusion through a C-terminal glycine-rich domain. Some splicing factors can regulate alternative use of splice site (ss) by binding to regulatory sequences between the two alternative sites. For example, ASF/5F2 can recognize ESEs and promote the use of intron proximal sites, whereas hnRNP Al can bind to ESSs and shift splicing towards the use of intron distal sites. One application for such factors is to generate ESFs that modulate alternative splicing of endogenous genes, particularly disease associated genes. For example, BCL2 like 1 (Bcl-x) pre-mRNA
produces two splicing isoforms with two alternative 5' splice sites to encode proteins of opposite functions. The long splicing isoform Bc1-xL is a potent apoptosis inhibitor expressed in long-lived post mitotic cells and is up-regulated in many cancer cells, protecting cells against apoptotic signals. The short isoform Bc1-xS is a pro-apoptotic isoform and expressed at high levels in cells with a high turnover rate (e.g., developing lymphocytes). The ratio of the two Bel-x splicing isoforms is regulated by multiple cc -elements that are located in either the core exon region or the exon extension region (i.e., between the two alternative 5' splice sites). For more examples, see W02010075303, which is hereby incorporated by reference in its entirety. Further suitable fusion partners include, but are not limited to proteins (or fragments thereof) that are boundary elements (e.g., CTCF), proteins and fragments thereof that provide periphery recruitment (e.g., Lamin A, Lamin B, etc.), and protein docking elements (e.g., FKBP/FRB, Pill/Abyl, etc.).

[00330] In some cases, a heterologous polypeptide (a fusion partner) for use with a reference CasX or CasX variant provides for subcellular localization, i.e., the heterologous polypeptide contains a subcellular localization sequence (e.g., a nuclear localization signal (NLS) for targeting to the nucleus, a sequence to keep the fusion protein out of the nucleus, e.g., a nuclear export sequence (NES), a sequence to keep the fusion protein retained in the cytoplasm, a mitochondrial localization signal for targeting to the mitochondria, a chloroplast localization signal for targeting to a chloroplast, an ER retention signal, and the like).
In some embodiments, a subject RNA-guided polypeptide or a conditionally active RNA-guided polypeptide and/or subject CasX fusion protein does not include a NLS so that the protein is not targeted to the nucleus, which can be advantageous; e.g., when the target nucleic acid is an RNA that is present in the cytosol. In some embodiments, a fusion partner can provide a tag (i.e., the heterologous polypeptide is a detectable label) for ease of tracking and/or purification (e.g., a fluorescent protein, e.g., green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), mCherry, tdTomato, and the like; a histidine tag, e.g., a 6XHis tag; a hemagglutinin (HA) tag; a FLAG tag; a Myc tag; and the like). In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4412-4415 and a subcellular localization sequence or a tag. In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 247-337, 3498-3501, 3505-3520, 3540-3549 and 4412-4415 and a subcellular localization sequence or a tag. In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 3498-3501, 3505-3520, and 3540-3549 and a subcellular localization sequence or a tag.

[00331] In some cases, a reference or CasX variant protein includes (is fused to) a nuclear localization signal (NLS). In some cases, a reference or CasX variant protein is fused to 2 or more, 3 or more, 4 or more, or 5 or more 6 or more, 7 or more, 8 or more NLSs.
In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the N-terminus and/or the C-terminus. In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the N-terminus. In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the C-terminus. In some cases, one or more NLSs (3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) both the N-terminus and the C-terminus. In some cases, an NLS is positioned at the N-terminus and an NLS is positioned at the C-terminus.
In some cases, a reference or CasX variant protein includes (is fused to) between 1 and 10 NLSs (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 2-10, 2-9, 2-8, 2-7, 2- 6, or 2-5 NLSs). In some cases, a reference or CasX variant protein includes (is fused to) between 2 and 5 NLSs (e.g., 2-4, or 2-3 NLSs).

[00332] Non-limiting examples of NLSs suitable for use with a reference CasX
or CasX variant include sequences having at least about 80%, at least about 90%, or at least about 95% identity or are identical to sequences derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 352); the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO:
353);
the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 354) or RQRRNELKRSP (SEQ ID NO: 355); the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 356); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO:
357) of the MB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO:
358) and PPKKARED (SEQ ID NO: 359) of the myoma T protein; the sequence PQPKKKPL
(SEQ
ID NO: 360) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 361) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 362) and PKQKKRK (SEQ ID NO: 363) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO: 364) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 365) of the mouse Mxl protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 366) of the human poly(ADP-ribose) polymerase; the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 367) of the steroid hormone receptors (human) glucocorticoid; the sequence PRPRKIPR (SEQ ID NO:
368) of Boma disease virus P protein (BDV-P1); the sequence PPRKKRTVV (SEQ ID NO: 369) of hepatitis C virus nonstructural protein (HCV-NS5A);the sequence NLSKKKKRKREK
(SEQ ID
NO: 370) of LEF1; the sequence RRPSRPFRKP (SEQ ID NO: 371) of 0RF57 simirae;
the sequence KRPRSPSS (SEQ ID NO: 372) of EBV LANA; the sequence KRGINDRNFWRGENERKTR (SEQ ID NO: 373) of Influenza A protein; the sequence PRPPKMARYDN (SEQ ID NO: 374) of human RNA helicase A (RHA); the sequence KRSFSKAF (SEQ ID NO: 375) of nucleolar RNA helicase II; the sequence KLKIKRPVK
(SEQ
ID NO: 376) of TUS-protein; the sequence PKKKRKVPPPPAAKRVKLD (SEQ ID NO: 377) associated with importin-alpha; the sequence PKTRRRPRRSQRKRPPT (SEQ ID NO:
378) from the Rex protein in HTLV-1; the sequence SRRRKANPTKLSENAKKLAKEVEN (SEQ ID
NO: 379) from the EGL-13 protein of Caenorhabditis elegans; and the sequences KTRRRPRRSQRKRPPT (SEQ ID NO: 380), RRKKRRPRRKKRR (SEQ ID NO: 381), PKKKSRKPKKKSRK (SEQ ID NO: 382), HKKKHPDASVNFSEFSK (SEQ ID NO: 383), QRPGPYDRPQRPGPYDRP (SEQ ID NO: 384), LSPSLSPLLSPSLSPL (SEQ ID NO: 385), RGKGGKGLGKGGAKRHRK (SEQ ID NO: 386), PKRGRGRPKRGRGR (SEQ ID NO: 387), PKKKRKVPPPPAAKRVKLD (SEQ ID NO: 388) and PKKKRKVPPPPKKKRKV (SEQ ID
NO: 389). In general, NLS (or multiple NLSs) are of sufficient strength to drive accumulation of a reference or CasX variant fusion protein in the nucleus of a eukaryotic cell. Detection of accumulation in the nucleus may be performed by any suitable technique. For example, a detectable marker may be fused to a reference or CasX variant fusion protein such that location within a cell may be visualized. Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation in the nucleus may also be determined indirectly.

[00333] In some embodiments, a CasX variant comprising an N terminal NLS
comprises a sequence of any one of SEQ ID NOS: 3508-3540-3549. In some embodiments, a CasX
variant comprising an N terminal NLS comprises a sequence with one or more additional modifications to of any one of SEQ ID NOS: 3508-3540-3549.

[00334] In some cases, a reference or CasX variant fusion protein includes a "Protein Transduction Domain" or PTD (also known as a CPP - cell penetrating peptide), which refers to a protein, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A
PTD attached to another molecule, which can range from a small polar molecule to a large macromolecule and/or a nanoparticle, facilitates the molecule traversing a membrane, for example going from an extracellular space to an intracellular space, or from the cytosol to within an organelle. In some embodiments, a PTD is covalently linked to the amino terminus of a reference or CasX variant fusion protein. In some embodiments, a PTD is covalently linked to the carboxyl terminus of a reference or CasX variant fusion protein. In some cases, the PTD is inserted internally in the sequence of a reference or CasX variant fusion protein at a suitable insertion site. In some cases, a reference or CasX variant fusion protein includes (is conjugated to, is fused to) one or more PTDs (e.g., two or more, three or more, four or more PTDs). In some cases, a PTD includes one or more nuclear localization signals (NLS). Examples of PTDs include but are not limited to peptide transduction domain of HIV TAT
comprising YGRKKRRQRRR (SEQ ID NO: 390), RKKRRQRR (SEQ ID NO: 391); YARAAARQARA
(SEQ ID NO: 392); THRLPRRRRRR (SEQ ID NO: 393); and GGRRARRRRRR (SEQ ID NO:
394); a polyarginine sequence comprising a number of arginines sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); an Drosophila Antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52(7): 1732-1737); a truncated human calcitonin peptide (Trehin et al.
(2004) Pharm. Research 21:1248-1256); polylysine (Wender et al. (2000) Proc.
Natl. Acad. Sci.
USA 97: 13003-13008); RRQRRTSKLMKR (SEQ ID NO: 395); Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 396);
KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO: 397); and RQIKIWFQNRRMKWKK (SEQ ID NO: 398). In some embodiments, the PTD is an activatable CPP (ACPP) (Aguilera et al. (2009) Integr Biol (Camb) June; 1(5-6): 371-381).
ACPPs comprise a polycationic CPP (e.g., Arg9 or "R9") connected via a cleavable linker to a matching polyanion (e.g., Glu9 or "E9"), which reduces the net charge to nearly zero and thereby inhibits adhesion and uptake into cells. Upon cleavage of the linker, the polyanion is released, locally unmasking the polyarginine and its inherent adhesiveness, thus "activating"
the ACPP to traverse the membrane. In some embodiments, a CasX variant comprises any one of SEQ ID
NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4412-4415 and a PTD. In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 247-337, 3498-3501, 3505-3520, 3540-3549 and 4412-4415 and a PTD. In some embodiments, a CasX
variant comprises any one of SEQ ID NOS: 3498-3501, 3505-3520, and 3540-3549 and a PTD.

[00335] In some embodiments, a reference or CasX variant fusion protein can include a CasX
protein that is linked to an internally inserted heterologous amino acid or heterologous polypeptide (a heterologous amino acid sequence) via a linker polypeptide (e.g., one or more linker polypeptides). In some embodiments, a reference or CasX variant fusion protein can be linked at the C-terminal and/or N-terminal end to a heterologous polypeptide (fusion partner) via a linker polypeptide (e.g., one or more linker polypeptides) The linker polypeptide may have any of a variety of amino acid sequences. Proteins can be joined by a spacer peptide, generally of a flexible nature, although other chemical linkages are not excluded.
Suitable linkers include polypeptides of between 4 amino acids and 40 amino acids in length, or between 4 amino acids and 25 amino acids in length. These linkers are generally produced by using synthetic, linker-encoding oligonucleotides to couple the proteins. Peptide linkers with a degree of flexibility can be used. The linking peptides may have virtually any amino acid sequence, bearing in mind that the preferred linkers will have a sequence that results in a generally flexible peptide. The use of small amino acids, such as glycine and alanine, are of use in creating a flexible peptide. The creation of such sequences is routine to those of skill in the art. A variety of different linkers are commercially available and are considered suitable for use. Example linker polypeptides include glycine polymers (G)n, glycine-serine polymer (including, for example, (GS)n, GSGGSn (SEQ
ID NO: 399), GGSGGSn (SEQ ID NO: 400), and GGGSn (SEQ ID NO: 401), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, glycine-proline polymers, proline polymers and proline-alanine polymers. Example linkers can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 402), GGSGG
(SEQ ID NO:
403), GSGSG (SEQ ID NO: 404), GSGGG (SEQ ID NO: 405), GGGSG (SEQ ID NO: 406), GSSSG (SEQ ID NO: 407), GPGP (SEQ ID NO: 408), GGP, PPP, PPAPPA (SEQ ID NO:
409), PPPGPPP (SEQ ID NO: 410) and the like. The ordinarily skilled artisan will recognize that design of a peptide conjugated to any elements described above can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure.
V. gNA and CasX Protein Gene Editing Pairs

[00336] In other aspects, provided herein are compositions of a gene editing pair comprising a CasX protein and a guide NA, referred to herein as a gene editing pair. In certain embodiments, the gene editing pair comprises a CasX variant protein as described herein (e.g., any one of the sequences set forth in Tables 3, 8, 9, 10 and 12) or a reference CasX protein as described herein (e.g., SEQ ID NOS:1-3), while, the guide NA is a reference gRNA (SEQ ID NOS: 4-16) or a gNA variant as described herein (e.g., SEQ ID NOS: 2101-2280), or sequence variants having at least 60%, or at least 70%, at least about 80%, or at least about 90%, or at least about 95%
sequence identity thereto, wherein the gNA comprises a targeting sequence complementary to the target DNA. In those embodiments in which one component is a variant, the pair is referred to as a variant gene editing pair. In other embodiments, a gene editing pair comprises the CasX
protein, a first gNA (either a reference gRNA {SEQ ID NOS: 4-16} or a gNA
variant as described herein {e.g.., SEQ ID NOS: 2101-2280}) with a targeting sequence, and a second gNA variant or a second reference guide nucleic acid, wherein the second gNA
variant or the second reference guide nucleic acid has a targeting sequence complementary to a different or overlapping portion of the target DNA compared to the targeting sequence of the first gNA.

[00337] In some embodiments, the variant gene editing pair has one or more improved characteristics compared to a reference gene editing pair, wherein the reference gene editing pair comprises a CasX protein of SEQ ID NOS: 1-3, a different gNA, or both. For example, in some embodiments, the variant gene editing pair comprises a CasX variant protein, and the variant gene editing pair has one or more improved characteristics compared to a reference gene editing pair comprising a reference CasX protein. In other embodiments, the variant gene editing pair comprises a gNA variant, and the variant gene editing pair has one or more improved characteristics compared to a reference gene editing pair comprising a reference gRNA. In other embodiments, the variant gene editing pair comprises a gNA variant and a CasX
variant protein, and the variant gene editing pair has one or more improved characteristics compared to a reference gene editing pair comprising a reference CasX protein and a reference gRNA.

[00338] In some embodiments of the variant gene editing pairs provided herein, the CasX is a variant protein as described herein (e.g., the sequences set forth in Tables 3, 8, 9, 10 and 12 or sequence variants having at least 60%, or at least 70%, at least about 80%, or at least about 90%, or at least about 95%, or at least about 99% sequence identity to the listed sequences) while the gNA is a reference gRNA of SEQ ID NO: 5 or SEQ ID NO: 4. In some embodiments of the variant gene editing pairs provided herein, the CasX comprises a reference CasX protein of SEQ
ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 while the gNA variant is a sequence of SEQ ID
NOS:2101-2280, or sequence variants having at least 60%, or at least 70%, at least about 80%, or at least about 90%, or at least about 95% sequence identity to the listed sequences.

[00339] In some embodiments, the variant gene editing pair has one or more improved characteristics compared to a reference gene editing pair comprising a reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the variant gene editing pair has one or more improved characteristics compared to a reference gene editing pair comprising a reference gRNA of SEQ ID NO: 5 or SEQ ID NO: 4. In some embodiments, the variant gene editing pair has one or more improved characteristics compared to a reference gene editing pair comprising a reference CasX protein of SEQ ID NO: 1, SEQ ID NO:
2, or SEQ ID
NO: 3 and a reference gRNA of SEQ ID NO: 5 or SEQ ID NO: 4.

[00340] Exemplary improved characteristics, as described herein, may in some embodiments, and include improved CasX:gNA RNP complex stability, improved binding affinity between the CasX and gNA, improved kinetics of RNP complex formation, higher percentage of cleavage-competent RNP, improved RNP binding affinity to the target DNA, improved unwinding of the target DNA, increased editing activity, improved editing efficiency, improved editing specificity, increased activity of the nuclease, increased target strand loading for double strand cleavage, decreased target strand loading for single strand nicking, decreased off-target cleavage, improved binding of the non-target strand of DNA, or improved resistance to nuclease activity. In the foregoing embodiments, the improvement is at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, at least about 1000-fold, at least about 5000-fold, at least about 10,000-fold, or at least about 100,000-fold compared to the characteristic of a reference CasX protein and reference gNA pair. In other cases, the one or more of the improved characteristics may be improved about 1.1 to 100,00-fold, about 1.1 to 10,00-fold, about 1.1 to 1,000-fold, about 1.1 to 500-fold, about 1.1 to 100-fold, about 1.1 to 50-fold, about 1.1 to 20-fold, about 10 to 100,00-fold, about 10 to 10,00-fold, about 10 to 1,000-fold, about 10 to 500-fold, about 10 to 100-fold, about 10 to 50-fold, about 10 to 20-fold, about 2 to 70-fold, about 2 to 50-fold, about 2 to 30-fold, about 2 to 20-fold, about 2 to 10-fold, about 5 to 50-fold, about 5 to 30-fold, about 5 to 10-fold, about 100 to 100,00-fold, about 100 to 10,00-fold, about 100 to 1,000-fold, about 100 to 500-fold, about 500 to 100,00-fold, about 500 to 10,00-fold, about 500 to 1,000-fold, about 500 to 750-fold, about 1,000 to 100,00-fold, about 10,000 to 100,00-fold, about 20 to 500-fold, about 20 to 250-fold, about 20 to 200-fold, about 20 to 100-fold, about 20 to 50-fold, about 50 to 10,000-fold, about 50 to 1,000-fold, about 50 to 500-fold, about 50 to 200-fold, or about 50 to 100-fold, improved relative to a reference gene editing pair. In other cases, the one or more of the improved characteristics may be improved about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, 180-fold, 190-fold, 200-fold, 210-fold, 220-fold, 230-fold, 240-fold, 250-fold, 260-fold, 270-fold, 280-fold, 290-fold, 300-fold, 310-fold, 320-fold, 330-fold, 340-fold, 350-fold, 360-fold, 370-fold, 380-fold, 390-fold, 400-fold, 425-fold, 450-fold, 475-fold, or 500-fold or more improved relative to a reference gene editing pair.

[00341] In some embodiments, the variant gene editing pair comprises a gNA
variant comprising a sequence of any one of SEQ ID NOs: 2101-2280 and a reference CasX
protein comprising an amino acid sequence of SEQ ID NO: 1. In some embodiments, the variant gene editing pair comprises a gNA variant comprising a sequence of any one of SEQ
ID NOS: 2101-2280 and a CasX variant protein comprising a variant of the reference CasX
protein of SEQ ID
NO: 2. In some embodiments, the variant gene editing pair comprises a reference gRNA

comprising a sequence of SEQ ID NO: 5 or SEQ ID NO: 4 and a CasX variant protein comprising a variant of the reference CasX protein of SEQ ID NO: 2. In some embodiments, the CasX variant protein comprises a Y789T substitution of SEQ ID NO: 2; a deletion of P at position 793 of SEQ ID NO: 2, a Y789D substitution of SEQ ID NO: 2, a T725 substitution of SEQ ID NO: 2, a I546V substitution of SEQ ID NO: 2, a E552A substitution of SEQ ID NO: 2, a A636D substitution of SEQ ID NO: 2, a F5365 substitution of SEQ ID NO: 2, a substitution of SEQ ID NO: 2, a Y797L substitution of SEQ ID NO: 2, a L792G
substitution of SEQ ID NO: 2, a A739V substitution of SEQ ID NO: 2, a G791M substitution of SEQ ID NO:
2, an insertion of A at position 661 of SEQ ID NO: 2, a A788W substitution of SEQ ID NO: 2, a K390R substitution of SEQ ID NO: 2, a A7515 substitution of SEQ ID NO: 2, a substitution of SEQ ID NO: 2, a combination of 5794R and Y797L substitutions of SEQ ID NO:
2, an insertion of P at 696 of SEQ ID NO: 2, a combination of K416E and A708K
substitutions of SEQ ID NO: 2, an insertion of M at position 773 of SEQ ID NO: 2, a G695H
substitution of SEQ ID NO: 2, an insertion of AS at position 793 of SEQ ID NO: 2, an insertion of AS at position 795 of SEQ ID NO: 2, a C477R substitution of SEQ ID NO: 2, a C477K
substitution of SEQ ID NO: 2, a C479A substitution of SEQ ID NO: 2, a C479L substitution of SEQ ID NO: 2, a combination of an A708K substitution and a deletion of P at position 793 of SEQ ID NO: 2, a 155F substitution of SEQ ID NO: 2, a K21OR substitution of SEQ ID NO: 2, a substitution of SEQ ID NO: 2, a D23 1N substitution of SEQ ID NO: 2, a Q338E
substitution of SEQ ID NO: 2, a Q338R substitution of SEQ ID NO: 2, a L379R substitution of SEQ ID NO: 2, a K390R substitution of SEQ ID NO: 2, a L481Q substitution of SEQ ID NO: 2, a substitution of SEQ ID NO: 2, a D600N substitution of SEQ ID NO: 2, a T886K
substitution of SEQ ID NO: 2, a combination of a deletion of P at position 793] and a P793A5 substitution of SEQ ID NO: 2, a A739V substitution of SEQ ID NO: 2, a K460N substitution of SEQ ID NO: 2, a I199F substitution of SEQ ID NO: 2, a G492P substitution of SEQ ID NO: 2, a substitution of SEQ ID NO: 2, a R591I substitution of SEQ ID NO: 2, an insertion of AS at position 795 of SEQ ID NO: 2, an insertion of AS at position 796 of SEQ ID NO:
2, an insertion of L at position 889 of SEQ ID NO: 2, a E121D substitution of SEQ ID NO: 2, a substitution of SEQ ID NO: 2, a E712Q substitution of SEQ ID NO: 2, a K942Q
substitution of SEQ ID NO: 2, a E552K substitution of SEQ ID NO: 2, a K25Q substitution of SEQ
ID NO: 2, a N47D substitution of SEQ ID NO: 2, a combination Q367K and I425S substitutions of SEQ ID
NO: 2, an insertion of T at position 696 of SEQ ID NO: 2, a L685I substitution of SEQ ID NO:

2, a N880D substitution of SEQ ID NO: 2, a combination of a A708K
substitution, a deletion of P at position 793 and a A739V substitution of SEQ ID NO: 2, a Q102R
substitution of SEQ ID
NO: 2, a M734K substitution of SEQ ID NO: 2, a A7245 substitution of SEQ ID
NO: 2, a T704K substitution of SEQ ID NO: 2, a P224K substitution of SEQ ID NO: 2, a combination of Q338R and A339E substitutions of SEQ ID NO: 2, a combination of Q338R and substitutions of SEQ ID NO: 2, a K25R substitution of SEQ ID NO: 2, a M29E
substitution of SEQ ID NO: 2, a H152D substitution of SEQ ID NO: 2, a 5219R substitution of SEQ ID NO:
2,a E475K substitution of SEQ ID NO: 2, a combination of 5507G and G508R
substitutions of SEQ ID NO: 2, a g226R substitution of SEQ ID NO: 2, a A377K substitution of SEQ ID NO: 2, a E480K substitution of SEQ ID NO: 2, a K416E substitution of SEQ ID NO: 2, a substitution of SEQ ID NO: 2, a K767R substitution of SEQ ID NO: 2, a I7F
substitution of SEQ ID NO: 2, a m29R substitution of SEQ ID NO: 2, a H435R substitution of SEQ
ID NO: 2, a E385Q substitution of SEQ ID NO: 2, a E385K substitution of SEQ ID NO: 2, a substitution of SEQ ID NO: 2, a D4895 substitution of SEQ ID NO: 2, a D732N
substitution of SEQ ID NO: 2, a A739T substitution of SEQ ID NO: 2, a W885R substitution of SEQ ID NO: 2, a E53K substitution of SEQ ID NO: 2, a A238T substitution of SEQ ID NO: 2, a substitution of SEQ ID NO: 2, a E292K substitution of SEQ ID NO: 2, a Q628E
substitution of SEQ ID NO: 2, a combination of F556I + D646A+G695D+A7515+A820P substitutions of SEQ
ID NO: 2, a R388Q substitution of SEQ ID NO: 2, a combination of L491I and substitutions of SEQ ID NO: 2, a G791M substitution of SEQ ID NO: 2, a L792K
substitution of SEQ ID NO: 2, a L792E substitution of SEQ ID NO: 2, a M779N substitution of SEQ ID NO: 2, a G27D substitution of SEQ ID NO: 2, a combination of L379R and A708K
substitutions and a deletion of P at position 793 of SEQ ID NO: 2, a combination of C477K and substitutions and a deletion of P at position 793 of SEQ ID NO: 2, a combination of L379R, C477K and A708K substitutions and a deletion of P at position 793 of SEQ ID
NO: 2, a combination of L379R, A708K and A739V substitutions and a deletion of P at position 793 of SEQ ID NO: 2, a combination of C477K, A708K and A739V substitutions and a deletion of P at position 793 of SEQ ID NO: 2, a combination of L379R, C477K, A708K and A739V
substitutions and a deletion of P at position 793 of SEQ ID NO: 2, a K955R
substitution of SEQ
ID NO: 2, a 5867R substitution of SEQ ID NO: 2, a R693I substitution of SEQ ID
NO: 2, a F189Y substitution of SEQ ID NO: 2, a V635M substitution of SEQ ID NO: 2, a substitution of SEQ ID NO: 2, a E498K substitution of SEQ ID NO: 2, a E386R
substitution of SEQ ID NO: 2, a V254G substitution of SEQ ID NO: 2, a P793S substitution of SEQ ID NO: 2, a K188E substitution of SEQ ID NO: 2, a QT945KI substitution of SEQ ID NO: 2, a T620P
substitution of SEQ ID NO: 2, a T946P substitution of SEQ ID NO: 2, a TT949PP
substitution of SEQ ID NO: 2, a N952T substitution of SEQ ID NO: 2 or a K682E substitution of SEQ ID
NO: 2.

[00342] In some embodiments, the variant gene editing pair comprises a CasX
gRNA of SEQ
ID NO: 5 and a CasX variant protein comprising a combination of L379R and substitutions and a deletion of P at position 793 of SEQ ID NO: 2. In some embodiments, the variant gene editing pair comprises a reference CasX protein SEQ ID NO: 2 and sgNA scaffold variant of SEQ ID NO: 5.

[00343] In some embodiments of the sgNA: protein variant pairs of the disclosure, the CasX
variant protein is selected from the group consisting of: a CasX variant protein comprising a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of T620P of SEQ ID NO: 2; a CasX variant protein comprising a substitution of M771A of SEQ ID NO: 2; a CasX variant protein comprising a substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a substitution of D732N of SEQ ID NO: 2; a CasX variant protein comprising a substitution of W782Q of SEQ
ID NO: 2; a CasX variant protein comprising a substitution of M771Q of SEQ ID NO: 2; a CasX variant protein comprises a substitution of R458I and a substitution of A739V of SEQ
ID NO: 2; a CasX
variant protein comprising a substitution of L379R, a substitution ofA708K, a deletion of P at position 793 and a substitution of M771N of SEQ ID NO: 2; a CasX variant protein comprising a substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a substitution of A739T of SEQ ID NO: 2; a CasX variant protein comprising a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of D4895 of SEQ ID NO: 2; a CasX variant protein comprising a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of D732N of SEQ ID NO: 2; a CasX variant protein comprising a substitution of V711K of SEQ ID NO: 2; a CasX variant protein comprising a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of Y797L of SEQ ID NO: 2; a CasX variant protein comprising a substitution of L379R, a substitution of A708K and a deletion of P at position 793 of SEQ ID NO: 2; a CasX variant protein comprising a substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of M771N of SEQ ID NO: 2; a CasX variant protein comprising a substitution of A708K, a substitution of P at position 793 and a substitution of E386S of SEQ ID NO: 2; a CasX variant protein comprising a substitution of L379R, a substitution of C477K, a substitution of A708K and a deletion of P at position 793 of SEQ ID
NO: 2; a CasX variant protein comprising a substitution of L792D of SEQ ID NO:
2; a CasX
variant protein comprising a substitution of G791F of SEQ ID NO: 2; a CasX
variant protein comprising a substitution of A708K, a deletion of P at position 793 and a substitution of A739V
of SEQ ID NO: 2; a CasX variant protein comprising a substitution of L379, a substitution of A708K, a deletion of P at position 793 and a substitution of A739V of SEQ ID
NO: 2; a CasX
variant protein comprising a substitution of C477K, a substitution of A708K
and a substitution of P at position 793 of SEQ ID NO: 2; a CasX variant protein comprising a substitution of L249I
and a substitution of M771N of SEQ ID NO: 2; a CasX variant protein comprising a substitution of V747K of SEQ ID NO: 2; and a CasX variant protein comprises a substitution of L379R, a substitution of C477, a substitution of A708K, a deletion of P at position 793 and a substitution of M779N of SEQ ID NO: 2; and the sequence encoding the sgNA variant is selected from the group consisting of SEQ ID NO: 2104, SEQ ID NO: 2163, SEQ ID NO: 2107, SEQ ID
NO:
2164, SEQ ID NO: 2165, SEQ ID NO: 2166, SEQ ID NO: 2103, SEQ ID NO: 2167, SEQ
ID
NO: 2105, SEQ ID NO: 2108, SEQ ID NO: 2112, SEQ ID NO: 2160, SEQ ID NO: 2170, SEQ
ID NO: 2114, SEQ ID NO: 2171, SEQ ID NO: 2112, SEQ ID NO: 2173, SEQ ID NO:
2102, SEQ ID NO: 2174, SEQ ID NO: 2175, SEQ ID NO: 2109, SEQ ID NO: 2176, SEQ ID NO:

2238, or SEQ ID NO: 2239.

[00344] In some embodiments, the gene editing pair comprises a CasX selected from any one of CasX of sequence SEQ ID NO: 270, SEQ ID NO: 292, SEQ ID NO: 311, SEQ ID NO:
333, or SEQ ID NO: 336, and a gNA selected from any one of SEQ ID NOS: 2104, 2106, or 2238.

[00345] In some embodiments, the gene editing pair comprises a CasX variant selected from any one of SEQ ID NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4415. In some embodiments, the gene editing pair comprises a CasX variant selected from any one of 247-337, 3498-3501, 3505-3520, 3540-3549 and 4412-4415. In some embodiments, the gene editing pair comprises a CasX variant selected from any one of 3498-3501, 3505-3520, and 3540-3549.

[00346] In some embodiments, the gene editing pair comprises a CasX variant selected from any one of SEQ ID NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4415 and a gNA selected from the group consisting of any one of SEQ ID NOS:
412-3295. In some embodiments, the gene editing pair comprises a CasX variant selected from any one of 247-337, 3498-3501, 3505-3520, 3540-3549 and 4412-4415, and a gNA selected from the group consisting of any one of SEQ ID NOS: 412-3295. In some embodiments, the gene editing pair comprises a CasX variant selected from any one of 3498-3501, 3505-3520, and 3540-3549, and a gNA selected from the group consisting of any one of SEQ ID NOS: 412-3295.

[00347] In some embodiments, the gene editing pair comprises a CasX variant selected from any one of SEQ ID NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4415 and a gNA selected from the group consisting of any one of SEQ ID NOS:
2101-2280. In some embodiments, the gene editing pair comprises a CasX variant selected from any one of 247-337, 3498-3501, 3505-3520, 3540-3549 and 4412-4415, and a gNA selected from the group consisting of any one of SEQ ID NOS: 2101-2280. In some embodiments, the gene editing pair comprises a CasX variant selected from any one of 3498-3501, 3505-3520, and 3540-3549, and a gNA selected from the group consisting of any one of SEQ ID NOS: 2101-2280.

[00348] In some embodiments, the gene editing pair comprises a CasX variant selected from any one of SEQ ID NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4415 and a gNA selected from the group consisting of any one of SEQ ID NOS:
2236, 2237, 2238, 2241, 2244, 2248, 2249, and 2259-2280. In some embodiments, the gene editing pair comprises a CasX variant selected from any one of 247-337, 3498-3501, 3505-3520, 3540-3549 and 4412-4415, and a gNA selected from the group consisting of any one of SEQ
ID NOS:
2236, 2237, 2238, 2241, 2244, 2248, 2249, and 2259-2280.. In some embodiments, the gene editing pair comprises a CasX variant selected from any one of 3498-3501, 3505-3520, and 3540-3549, and a gNA selected from the group consisting of any one of SEQ ID
NOS: 2236, 2237, 2238, 2241, 2244, 2248, 2249, and 2259-2280.

[00349] In still further embodiments, the present disclosure provides a gene editing pair comprising a CasX protein and a gNA, wherein the gNA is a guide RNA variant as described herein. In some embodiments of the gene editing pairs of the disclosure, the Cas protein is a CasX variant as described herein. In some embodiments, the CasX protein is a reference CasX
protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 and the gNA is a guide RNA variant as described herein. Exemplary improved characteristics of the gene editing pair embodiments, as described herein, may in some embodiments include improved protein:gNA
complex stability, improved ribonuclear protein complex (RNP) formation, higher percentage of cleavage-competent RNP, improved binding affinity between the CasX protein and gNA, improved binding affinity to the target DNA, improved unwinding of the target DNA, increased activity, improved editing efficiency, improved editing specificity, increased activity of the nuclease, increased target strand loading for double strand cleavage, decreased target strand loading for single strand nicking, decreased off-target cleavage, improved binding of the non-target strand of DNA, or improved resistance to nuclease activity. In the foregoing embodiments, the improvement is at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, at least about 1000-fold, at least about 5000-fold, at least about 10,000-fold, or at least about 100,000-fold compared to the characteristic of a reference CasX protein and reference gNA pair.

[00350] In some embodiments, wherein the gene editing pair comprises both a CasX variant protein and a gNA variant as described herein, the one or more characteristics of the gene editing pair is improved beyond what can be achieved by varying the CasX protein or the gNA alone. In some embodiments, the CasX variant protein and the gNA variant act additively to improve one or more characteristics of the gene editing pair. In some embodiments, the CasX variant protein and the gNA variant act synergistically to improve one or more characteristics of the gene editing pair. In the foregoing embodiments, the improvement is at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, at least about 1000-fold, at least about 5000-fold, at least about 10,000-fold, or at least about 100,000-fold compared to the characteristic of a reference CasX protein and reference gNA pair.
VI. Methods of Making CasX Variant Protein and gNA Variants

[00351] The CasX variant proteins and gNA variants as described herein may be constructed through a variety of methods. Such methods may include, for example, Deep Mutational Evolution (DME), described below and in the Examples.
a. Deep Mutational Evolution (DME)

[00352] In some embodiments, DME is used to identify CasX protein and sgNA
scaffold variants with improved function. The DME method, in some embodiments, comprises building and testing a comprehensive set of mutations to a starting biomolecule to produce a library of biomolecule variants; for example, a library of CasX variant proteins or sgNA
scaffold variants.
DME can encompass making all possible substitutions, as well as all possible small insertions, and all possible deletions of amino acids (in the case of proteins) or nucleotides (in the case of RNA or DNA) to the starting biomolecule. A schematic illustrating DME methods is shown in FIG. 1. In some embodiments, DME comprises a subset of all such possible substitutions, insertions, and deletions. In certain embodiments of DME, one or more libraries of variants are constructed, evaluated for functional changes, and this information used to construct one or more additional libraries. Such iterative construction and evaluation of variants may lead, for example, to identification of mutational themes that lead to certain functional outcomes, such as regions of the protein or RNA that when mutated in a certain way lead to one or more improved functions. Layering of such identified mutations may then further improve function, for example through additive or synergistic interactions. DME comprises library design, library construction, and library screening. In some embodiments, multiple rounds of design, construction, and screening are undertaken.
b. Library Design

[00353] DME methods produce variants of biomolecules, which are polymers of many monomers. In some embodiments, the biomolecule comprises a protein or a ribonucleic acid (RNA) molecule, wherein the monomer units are amino acids or ribonucleotides, respectively.
The fundamental units of biomolecule mutation comprise either: (1) exchanging one monomer for another monomer of different identity (substitutions); (2) inserting one or more additional monomer in the biomolecule (insertions); or (3) removing one or more monomer from the biomolecule (deletions). DME libraries comprising substitutions, insertions, and deletions, alone or in combination, to any one or more monomers within any biomolecule described herein, are considered within the scope of the invention.

[00354] In some embodiments, DME is used to build and test the comprehensive set of mutations to a biomolecule, encompassing all possible substitutions, as well as small insertions and deletions of amino acids (in the case of proteins) or nucleotides (in the case of RNA). The construction and functional readout of these mutations can be achieved with a variety of established molecular biology methods. In some embodiments, the library comprises a subset of all possible modifications to monomers. For example, in some embodiments, a library collectively represents a single modification of one monomer, for at least 10%
of the total monomer locations in a biomolecule, wherein each single modification is selected from the group consisting of substitution, single insertion, and single deletion. In some embodiments, the library collectively represents the single modification of one monomer, for at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or up to 100% of the total monomer locations in a starting biomolecule. In certain embodiments, for a certain percentage of the total monomer locations in a starting biomolecule, the library collectively represents each possible single modification of a one monomer, such as all possible substitutions with the 19 other naturally occurring amino acids (for a protein) or 3 other naturally occurring ribonucleotides (for RNA), insertion of each of the 20 naturally occurring amino acids (for a protein) or 4 naturally occurring ribonucleotides (for RNA), or deletion of the monomer. In still further embodiments, insertion at each location is independently greater than one monomer, for example insertion of two or more, three or more, or four or more monomers, or insertion of between one to four, between two to four, or between one to three monomers. In some embodiments, deletion at location is independently greater than one monomer, for example deletion of two or more, three or more, or four or more monomers, or deletion of between one to four, between two to four, or between one to three monomers.
Examples of such libraries of CasX variants and gNA variants are described in Examples 24 and 25, respectively.

[00355] In some embodiments, the biomolecule is a protein and the individual monomers are amino acids. In those embodiments where the biomolecule is a protein, the number of possible DME mutations at each monomer (amino acid) position in the protein comprise 19 amino acid substitutions, 20 amino acid insertions and 1 amino acid deletion, leading to a total of 40 possible mutations per amino acid in the protein.

[00356] In some embodiments, a DME library of CasX variant proteins comprising insertions is 1 amino acid insertion library, a 2 amino acid insertion library, a 3 amino acid insertion library, a 4 amino acid insertion library, a 5 amino acid insertion library, a 6 amino acid insertion library, a 7 amino acid insertion library, an 8 amino acid insertion library, a 9 amino acid insertion library or a 10 amino acid insertion library. In some embodiments, a DME library of CasX variant proteins comprising insertions comprises between 1 and 4 amino acid insertions.

[00357] In some embodiments, the biomolecule is RNA. In those embodiments where the biomolecule is RNA, the number of possible DME mutations at each monomer (ribonucleotide) position in the RNA comprises 3 nucleotide substitutions, 4 nucleotide insertions, and 1 nucleotide deletion, leading to a total of 8 possible mutations per nucleotide.

[00358] In some embodiments, DME library design comprises enumerating all possible mutations for each of one or more target monomers in a biomolecule. As used herein, a "target monomer" refers to a monomer in a biomolecule polymer that is targeted for DME
with the substitutions, insertions and deletions described herein. For example, a target monomer can be an amino acid at a specified position in a protein, or a nucleotide at a specified position in an RNA. A biomolecule can have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100 or more target monomers that are systematically mutated to produce a DME library of biomolecule variants. In some embodiments, every monomer in a biomolecule is a target monomer. For example, in DME of a protein where there are two target amino acids, DME
library design comprises enumerating the 40 possible DME mutations at each of the two target amino acids. In a further example, in DME of an RNA where there are four target nucleotides, DME library design comprises enumerating the 8 possible DME mutations at each of the four target nucleotides. In some embodiments, each target monomer of a biomolecule is independently randomly selected or selected by intentional design. Thus, in some embodiments, a DME library comprises random variants, or variants that were designed, or variants comprising random mutations and designed mutations within a single biomolecule, or any combinations thereof.

[00359] In some embodiments of DME methods, DME mutations are incorporated into double-stranded DNA encoding the biomolecule. This DNA can be maintained and replicated in a standard cloning vector, for example a bacterial plasmid, referred to herein as the target plasmid.
An exemplary target plasmid contains a DNA sequence encoding the starting biomolecule that will be subjected to DME, a bacterial origin of replication, and a suitable antibiotic resistance expression cassette. In some embodiments, the antibiotic resistance cassette confers resistance to kanamycin, ampicillin, spectinomycin, bleomycin, streptomycin, erythromycin, tetracycline or chloramphenicol. In some embodiments, the antibiotic resistance cassette confers resistance to kanamycin.

[00360] A library comprising said variants can be constructed in a variety of ways. In certain embodiments, plasmid recombineering is used to construct a library. Such methods can use DNA oligonucleotides encoding one or more mutations to incorporate said mutations into a plasmid encoding the reference biomolecule. For biomolecule variants with a plurality of mutations, in some embodiments more than one oligonucleotide is used. In some embodiments, the DNA oligonucleotides encoding one or more mutations wherein the mutation region is flanked by between 10 and 100 nucleotides of homology to the target plasmid, both 5' and 3' to the mutation. Such oligonucleotides can in some embodiments be commercially synthesized and used in PCR amplification. An exemplary template for an oligonucleotide encoding a mutation is provided below:

'- (N)io-loo - Mutation ¨ (N')io-ioo - 3'

[00361] In this exemplary oligonucleotide design, the Ns represent a sequence identical to the target plasmid, referred to herein as the homology arms. When a particular monomer in the biomolecule is targeted for mutation, these homology arms directly flank the DNA encoding the monomer in the target plasmid. In some exemplary embodiments where the biomolecule undergoing DME is a protein, 40 different oligonucleotides, using the same set of homology arms, are used to encode the enumerated 40 different amino acid mutations for each amino acid residue in the protein that is targeted for DME. When the mutation is of a single amino acid, the region encoding the desired mutation or mutations comprises three nucleotides encoding an amino acid (for substitutions or single insertions), or zero nucleotides (for deletions). In some embodiments, the oligonucleotide encodes insertion of greater than one amino acid. For example, wherein the oligonucleotide encodes the insertion of X amino acids, the region encoding the desired mutation comprises 3*X nucleotides encoding the X amino acids. In some embodiments, the mutation region encodes more than one mutation, for example mutations to two or more monomers of a biomolecule that are in close proximity (e.g., next to each other, or within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more monomers of each other).

[00362] Nucleotide sequences code for particular amino acid monomers in a substitution or insertion mutation in an oligo as described herein will be known to the person of ordinary skill in the art. For example, TTT or TTC triplets can be used to encode phenylalanine;
TTA, TTG, CTT, CTC, CTA or CTG can be used to encode leucine; ATT, ATC or ATA can be used to encode isoleucine; ATG can be used to encode methionine; GTT, GTC, GTA or GTG
c can be used to encode valine; TCT, TCC, TCA, TCG, AGT or AGC can be used to encode serine;
CCT, CCC, CCA or CCG can be used to encode proline; ACT, ACC, ACA or ACG can be used to encode threonine; GCT, GCC, GCA or GCG can be used to encode alanine; TAT
or TAC can be used to encode tyrosine; CAT or CAC can be used to encode histidine; CAA or CAG can be used to encode glutamine, AAT or AAC can be used to encode asparagine; AAA or AAG can be used to encode lysine; GAT or GAC can be used to encode aspartic acid; GAA or GAG can be used to encode glutamic acid; TGT or TGC c can be used to encode cysteine; TGG
can be used to encode tryptophan; CGT, CGC, CGA, CGG, AGA or AGG can be used to encode arginine;
and GGT, GGC, GGA or GGG can be used to encode glycine. In addition, ATG is used for initiation of the peptide synthesis as well as for methionine and TAA, TAG and TGA can be used to encode for the termination of the peptide synthesis.

[00363] In some exemplary embodiments where the biomolecule undergoing DME is an RNA, 8 different oligonucleotides, using the same set of homology arms, encode the above enumerated 8 different single nucleotide mutations for each nucleotide in the RNA that is targeted for DME.
When the mutation is of a single ribonucleotide, the region of the oligo encoding the mutations can consist of the following nucleotide sequences: one nucleotide specifying a nucleotide (for substitutions or insertions), or zero nucleotides (for deletions). In some embodiments, the oligonucleotides are synthesized as single stranded DNA oligonucleotides. In some embodiments, all oligonucleotides targeting a particular amino acid or nucleotide of a biomolecule subjected to DME are pooled. In some embodiments, all oligonucleotides targeting a biomolecule subjected to DME are pooled. There is no limit to the type or number of mutations that can be created simultaneously in a DME library.
c. DME Library Construction

[00364] In some embodiments, plasmid recombineering is utilized to construct one or more DME libraries. Plasmid recombineering is described in Higgins, Sean A., Sorel V. Y. Ouonkap, and David F. Savage (2017) "Rapid and Programmable Protein Mutagenesis Using Plasmid Recombineering" ACS Synthetic Biology, the contents of which are incorporated herein by reference in their entirety.

[00365] An exemplary library construction protocol shown below:

[00366] Day 1: A bla, bio-, lambda-Red 1, mutS¨, cmR E. coli strain (for example, EcNR2, Addgene ID: 26931) is streaked out on a LB agar plate containing standard concentrations of the antibiotics Chloramphenicol and Ampicillin. Colonies are grown overnight at 30 C.

[00367] Day 2: A single colony of EcNR2 is picked into 5 mL of LB liquid media containing standard concentrations of the antibiotics Chloramphenicol and Ampicillin. The culture is grown overnight with shaking at 30 C.

[00368] Day 3: Electrocompetent cells are made using any method known in the art. An non-limiting, exemplary protocol for making electrocompetent cells comprises:
(1) Dilute 50 uL of the overnight culture into 50 mL of LB liquid media containing standard concentrations of the antibiotics Chloramphenicol and Ampicillin.
Grow this 50 mL
culture with shaking at 30 C.
(2) Once the 50 mL culture has grown to an 0D600 = 0.5, transfer to shaking growth at 42 C in a liquid water bath. Care should be taken to limit this growth at 42 C to 15 minutes.

(3) After heated growth, transfer the culture to an ice water bath and swirl for at least one minute to cool the culture.
(4) Pellet the culture by spinning at 4,000 x g for 10 minutes. Decant the supernatant.
(5) Carefully wash and re-suspend the pellet by adding ice cold water up to 50 mL.
Repeat spin step 4.
(6) Resuspend the pellet in 1 mL of ice cold water. The cells are now competent for a standard electroporation step.

[00369] The electrocompetent E. coil are then transformed with the DME
oligonucleotides:
(1) Pooled DME oligonucleotides are diluted in water to a final concentration of 20 [NI.
If more than one mutation is to be generated simultaneously, the corresponding oligonucleotides should be combined and mixed thoroughly.
(2) Pure target plasmid, for example, from a miniprep, is diluted in water to a final concentration of 10 ng per [IL.
(3) Mix on ice:
2.5 [IL DME oligonucleotide mixture 1 pL target plasmid 46.5 [IL electrocompetent EcNR2 cells (4) Transfer the mixture to a sterile 0.1 cm electroporation cuvette on ice and perform an electroporation. For example, the parameters of 1800 kV, 200 S2, 25 g can be used.
(5) Recover the electroporated cells by adding 1 mL of standard warm SOC
media. Grow the culture for one hour with shaking at 30 C.
(6) After the recovery, add 4 mL of additional standard LB media to the culture. Add Kanamycin antibiotic at standard concentrations in order to select for the electroporated target plasmid. The culture is then grown =overnight with shaking at 30 C.

[00370] Day 4. Methods of isolating the target plasmid from overnight cultures will be readily apparent to one of ordinary skill in the art. For example, target plasmid can be isolated using commercial MiniPrep kits such as the MiniPrep kit from Qiagen. The plasmid library obtained comprises mutated target plasmids. In some embodiments, the plasmid library comprises between 10% and 30% mutated target plasmids. Additional mutations can be progressively added by repeatedly passing the library through rounds of electroporation and outgrowth, with no practical limit on the number of rounds that may be performed. Thus, for example, in some embodiments the library comprises plasmids encoding greater than one mutation per plasmid.

For example, in some embodiments the library comprises plasmids independently comprising one, two, three, four, five, six, seven eight, nine, or greater mutations per plasmid. In some embodiments, plasmids that do not comprise any mutations are also present (e.g., plasmids which did not incorporate a DME oligonucleotide).

[00371] In other embodiments, methods other than plasmid recombineering are used to construct one or more DME libraries, or a combination of plasmid recombineering and other methods are used to construct one or more DME libraries. For example, DME
libraries may, in some embodiments, be constructed using one of the other mutational methods described herein.
Such libraries may then be taken through the library screening as described herein, and further iterations be carried out if desired.
d. Library Screening

[00372] Any appropriate method for screening or selecting a DME library is envisaged as following within the scope of the inventions. High throughput methods may be used to evaluate large libraries with thousands of individual mutations. In some embodiments, the throughput of the library screening or selection assay has a throughput that is in the millions of individual cells.
In some embodiments, assays utilizing living cells are preferred, because phenotype and genotype are physically linked in living cells by nature of being contained within the same lipid bilayer. Living cells can also be used to directly amplify sub-populations of the overall library.
In other embodiments, smaller assays are used in DME methods, for example to screen a focused library developed through multiple rounds of mutation and evaluation.
Exemplary methods of screening libaries are described in Examples 24 and 25.

[00373] An exemplary, but non-limiting DME screening assay comprises Fluorescence-Activated Cell Sorting (FACS). In some embodiments, FACS may be used to assay millions of unique cells in a DME library. An exemplary FACS screening protocol comprises the following steps:
(1) PCR amplifying the purified plasmid library from the library construction phase.
Flanking PCR primers can be designed that add appropriate restriction enzyme sites flanking the DNA encoding the biomolecule. Standard oligonucleotides can be used as PCR
primers, and can be synthesized commercially. Commercially available PCR reagents can be used for the PCR
amplification, and protocols should be performed according to the manufacturer's instructions.
Methods of designing PCR primers, choice of appropriate restriction enzyme sites, selection of PCR reagents and PCR amplification protocols will be readily apparent to the person of ordinary skill in the art.
(2) The resulting PCR product is digested with the designed flanking restriction enzymes.
Restriction enzymes may be commercially available, and methods of restriction enzyme digestion will be readily apparent to the person of ordinary skill in the art.
(3) The PCR product is ligated into a new DNA vector. Appropriate DNA vectors may include vectors that allow for the expression of the DME library in a cell.
Exemplary vectors include, but are not limited to, retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral (AAV) vectors and plasmids. This new DNA vector can be part of a protocol such as lentiviral integration in mammalian tissue culture, or a simple expression method such as plasmid transformation in bacteria. Any vectors that allow for the expression of the biomolecule, and the DME library of variants thereof, in any suitable cell type, are considered within the scope of the disclosure. Cell types may include bacterial cells, yeast cells, and mammalian cells.
Exemplary bacterial cell types may include E. coil. Exemplary yeast cell types may include Saccharomyces cerevisiae. Exemplary mammalian cell types may include mouse, hamster, and human cell lines, such as HEK293 cells, HEK293T cells, HEK293-F cells, Lenti-X
293T cells, BHK cells, HepG2 cells, Saos-2 cells, HuH7 cells, A549 cells, NSO cells, SP2/0 cells, YO
myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, hybridoma cells, VERO
cells, NIH3T3 cells, COS, WI38 cells, MRCS cells, HeLa, HT1080 cells, or CHO
cells.. Choice of vector and cell type will be readily apparent to the person of ordinary skill in the art. DNA
ligase enzymes can be purchased commercially, and protocols for their use will also be readily apparent to one of ordinary skill in the art.
(4) Once the DME library has been cloned into a vector suitable for in vivo expression, the DME library is screened. If the biomolecule has a function which alters fluorescent protein production in a living cell, the biomolecule's biochemical function will be correlated with the fluorescence intensity of the cell overall. By observing a population of millions of cells on a flow cytometer, a DME library can be seen to produce a broad distribution of fluorescence intensities. Individual sub-populations from this overall broad distribution can be extracted by FACS. For example, if the function of the biomolecule is to repress expression of a fluorescent protein, the least bright cells will be those expressing biomolecules whose function has been improved by DME. Alternatively, if the function of the biomolecule is to increase expression of a fluorescent protein, the brightest cells will be those expressing biomolecules whose function has been improved by DME. Cells can be isolated based on fluorescence intensity by FACS and grown separately from the overall population. An exemplary FACS screening assay is shown in FIG. 2.
(5) After FACS sorting cells expressing a DME library of biomolecule variants, cultures comprising the original DME library and/or only highly functional biomolecule variants, as determined by FACS sorting, can be amplified separately. If the cells that were FACS sorted comprise cells that express the DME library of biomolecule variants from a plasmid (for example, E. coil cells transformed with a plasmid expression vector), these plasmids can be isolated, for example through miniprep. Conversely if the DME library of biomolecule variants has been integrated into the genomes of the FACs sorted cells, this DNA region can be PCR
amplified and, optionally, subcloned into a suitable vector for further characterization using methods known in the art. Thus, the end product of library screening is a DNA
library representing the initial, or 'naive', DME library, as well as one or more DNA
libraries containing sub-populations of the naive DME library, which comprise highly functional mutant variants of the biomolecule identified by the screening processes described herein.

[00374] In some embodiments, DME libraries that have been screened or selected for highly functional variants are further characterized. In some embodiments, further characterizing the DME library comprises analyzing DME variants individually through sequencing, such as Sanger sequencing, to identify the specific mutation or mutations that gave rise to the highly functional variant. Individual mutant variants of the biomolecule can be isolated through standard molecular biology techniques for later analysis of function. In some embodiments, further characterizing the DME library comprises high throughput sequencing of both the naive library and the one or more libraries of highly functional variants. This approach may, in some embodiments, allow for the rapid identification of mutations that are over-represented in the one or more libraries of highly functional variants compared to the naïve DME
library. Without wishing to be bound by any theory, mutations that are over-represented in the one or more libraries of highly functional variants are likely to be responsible for the activity of the highly functional variants. In some embodiments, further characterizing the DME
library comprises both sequencing of individual variants and high throughput sequencing of both the naive library and the one or more libraries of highly functional variants.

[00375] High throughput sequencing can produce high throughput data indicating the functional effect of the library members. In embodiments wherein one or more libraries represents every possible mutation of every monomer location, such high throughput sequencing can evaluate the functional effect of every possible DME mutation. Such sequencing can also be used to evaluate one or more highly functional sub-populations of a given library, which in some embodiments may lead to identification of mutations that result in improved function. An exemplary protocol for high throughput sequencing of a library with a highly functional sub-population is as follows:
(1) High throughput sequencing of the Naive DME library, N. High throughput sequence the highly functional sub-population library, F. Any high throughput sequencing platform that can generate a suitable abundance of reads can be used. Exemplary sequencing platforms include, but are not limited to Illumina, Ion Torrent, 454 and PacBio sequencing platforms.
(2) Select a particular mutation to evaluate, i. Calculate the total fractional abundance of i in N, i(N). Calculate the total fractional abundance of i in F, i(F).
(3) Calculate the following: [ ( i(F) + 1) / ( i(N) + 1 ) ]. This value, the 'enrichment ratio', is correlated with the function of the particular mutant variant i of the biomolecule.
(4) Calculate the enrichment ratio for each of the mutations observed in deep sequencing of the DME libraries.
(5) The set of enrichment ratios for the entire library can be converted to a log scale such that a value of zero represents no enrichment (i.e. an enrichment ratio of one), values greater than zero represent enrichment, and values less than zero represent depletion.
Alternatively, the log scale can be set such that 1.5 represents enrichment, and -0.6 represents depletion, as in FIG.
3A, FIG. 3B, FIG. 4A, FIG. 4C. These rescaled values can be referred to as the relative 'fitness' of any particular mutation. These fitness values quantitatively indicate the effect a particular mutation has on the biochemical function of the biomolecule.
(6) The set of calculated DME fitness values can be mapped to visually represent the fitness landscape of all possible mutations to a biomolecule. The fitness values can also be rank ordered to determine the most beneficial mutations contained within the DME
library.
e. Iterating DME

[00376] In some embodiments, a highly functional variant produced by DME has more than one mutation. For example, combinations of different mutations can in some embodiments produce optimized biomolecules whose function is further improved by the combination of mutations. In some embodiments, the effect of combining mutations on function of the biomolecule is linear. As used herein, a combination of mutations that is linear refers to a combination whose effect on function is equal to the sum of the effects of each individual mutation when assayed in isolation. In some embodiments, the effect of combining mutations on function of the biomolecule is synergistic. As used herein, a combination of mutations that is synergistic refers to a combination whose effect on function is greater than the sum of the effects of each individual mutation when assayed in isolation. Other mutations may exhibit additional unexpected nonlinear additive effects, or even negative effects. This phenomenon is known as epistasis.

[00377] Epistasis can be unpredictable, and is a significant source of variation when combining mutations. Epistatic effects can be addressed through additional high throughput experimental methods in DME library construction and assay. In some embodiments, the entire DME protocol can be iterated, returning to the library construction step and selecting only mutations identified as having desired effects (such as increased functionality) from an initial DME library screen.
Thus, in some embodiments, DME library construction and screening is iterated, with one or more cycles focusing the library on a subset of mutations having desired effects. In such embodiments, layering of selected mutations may lead to improved variants. In some alternative embodiments, DME can be repeated with the full set of mutations, but targeting a novel, pre-mutated version of the biomolecule. For example, one or more highly functional variants identified in a first round of DME library construction, assay, and characterization can be used as the target plasmid for further rounds of DME using a broad, unfocused set of further mutations (such as every possible mutation, or a subset thereof), and the process repeated. Any number, type of iterations or combinations of iterations of DME are envisaged as within the scope of the disclosure.
f. Deep Mutational Scanning

[00378] In some embodiments, Deep Mutational Scanning (DMS) is used to identify CasX
variant proteins with improved function. Deep mutational scanning assesses protein plasticity as it relates to function. In DMS methods, every amino acid of a protein is changed to every other amino acid and absolute protein function assayed. For example, every amino acid in a CasX
protein can be changed to every other amino acid, and the mutated CasX
proteins assayed for their ability to bind to or cleave DNA. Exemplary assays such as the CRISPRi assay or bacterial-based cleavage assays that can be used to characterize collections of DMS CasX
variant proteins are described in Oakes et al. (2016) "Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch" Nat Biotechnol 34(6):646-51 and Liu et al. (2019) "CasX
enzymes comprise a distinct family of RNA-guided genome editors" Nature doi.org/10.1038/s41586-019-0908; the contents of which are incorporated herein by reference.

[00379] In some embodiments, DMS is used to identify CasX proteins with improved DNA
binding activity. In some embodiments, DNA binding activity is assayed using a CRISPRi assay. In a non-limiting, exemplary embodiment of a CRISPRi assay, cells expressing a fluorescent protein such as green fluorescent protein (GFP) or red fluorescent protein (RFP) are assayed using FACS to identify CasX variants capable of repressing expression of the fluorescent protein in a sgNA dependent fashion. In this example, a catalytically dead CasX
(dCasX) is used to generate the collection of DMS mutants being assayed. The wild-type CasX
protein binds to its cognate sgNA and forms a protein-RNA complex. The complex binds to specific DNA targets by Watson-Crick base pairing between the sgNA and the DNA
target, in this case a DNA sequence encoding the fluorescent protein. In the case of wild-type CasX, the DNA will be cleaved due to the nuclease activity of the CasX protein. However, without wishing to be bound by theory, it is likely that dCasX is still able to form a complex with the sgNA and bind to specific DNA target. When targeting of dCasX occurs to the protein-coding region, it blocks RNA polymerase II and transcript initiation and/or elongation, leading to a reduction in fluorescent protein expression that can be detected by FACs.

[00380] In some embodiments, DMS is used to identify CasX proteins with improved DNA
cleavage activity. Methods of assaying the DNA cleavage efficiency of CasX
variant proteins will be apparent to one of ordinary skill in the art. For example, CasX
proteins complexed with an sgNA with a spacer complementary to a particular target DNA sequence can be used to cleave the DNA target sequence in vitro or in vivo in a suitable cell type, and the frequency of insertions and deletions at the site of cleavage are assayed. Without wishing to be bound by theory, cleavage or nicking by CasX generates double-strand breaks in DNA, whose subsequent repair by the non-homologous end joining pathway (NHEJ) gives rise to small insertions or deletions (indels) at the site of the double-strand breaks. The frequency of indels at the site of CasX cleavage can be measured using high throughput or Sanger sequencing of the target sequence. Alternatively, or in addition, frequency of indel generation by CasX
cleavage of a target sequence can be measured using mismatch assays such as T7 Endonuclease I (T7EI) or Surveyor mismatch assays.

[00381] In some embodiments, following DMS, a map of the genotypes of DMS
mutants linked with their resulting phenotype (for example, a heat map) is generated and used to characterize fundamental principles of the protein. All possible mutations are characterized as leading to functional or nonfunctional protein products to establish that protein's functional landscape.
g. Error Prone PCR

[00382] In some embodiments, Error Prone PCR is used to generate CasX protein or sgNA
scaffold variants with improved function. Polymerases that replicate DNA have different levels of fidelity. One way of introducing random mutations to a gene is through an error prone polymerase that will incorporate incorrect nucleotides at a range of frequencies. This frequency can be modulated depending on the desired outcome. In some embodiments, a polymerase and conditions for polymerase activity are selected that result in a frequency of nucleotide changes that produces an average of n 1-4 amino acid changes in a protein sequence. An exemplary error prone polymerase comprises Agilent's GeneMorphII kit. The GeneMorphII kit can be used to amplify a DNA sequence encoding a wild type CasX protein (for example, a protein of SEQ ID
NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3), according to the manufacturer's protocol, thereby subjecting the protein to unbiased random mutagenesis and generating a diverse population of CasX variant proteins. This diverse population of CasX variant proteins can then be assayed using the same assays described above for DMS to observe how changes in genotype relate to changes in phenotype.
h. Cassette Mutagenesis

[00383] In some embodiments, cassette mutagenesis is used to generate CasX
variant protein or sgNA scaffold variants with improved function. Cassette mutagenesis takes advantage of unique restriction enzyme sites that are replaced by degenerative nucleotides to create small regions of high diversity in select areas of a gene of interest such as a CasX protein or sgNA scaffold. In an exemplary cassette mutagenesis protocol, restriction enzymes are used to cleave near the sequence targeted for mutagenesis on DNA molecule encoding a CasX protein or sgNA scaffold contained in a suitable vector. This step removes the sequence targeted for mutagenesis and everything between the restriction sites. Then, synthetic double stranded DNA
molecules containing the desired mutation and ends that are complimentary to the restriction digest ends are ligated in place of the sequence that has been removed by restriction digest, and suitable cells, such as E. coil are transformed with the ligated vector. In some embodiments, cassette mutagenesis can be used to generate one or more specific mutations in a CasX
protein or sgNA
scaffold. In some embodiments, cassette mutagenesis can be used to generate a library of CasX
variant proteins or sgNA scaffold variants that can be screened or selected for improved function using the methods described herein. For example, in using cassette mutagenesis to generate CasX variants, parts of the Non-Target Strand Binding (NTSB) domain can be replaced with a sequence of degenerate nucleotides. Sequences of degenerate nucleotides can be highly localized to regions of the CasX protein, for example regions of the NTSB that are of interest because of their highly mobile elements or their direct contacts with DNA. Libraries of CasX variant proteins generated via cassette mutagenesis can then be screened using the assays described herein for DME, DMS and error prone PCR and variants can be selected for improved function.
i. Random Mutagenesis

[00384] In some embodiments, random mutagenesis is used to generate CasX
variant proteins or sgNA scaffold variants with improved function. Random mutagenesis is an unbiased way of changing DNA. Exemplary methods of random mutagenesis will be known to the person of ordinary skill in the art and include exposure to chemicals, UV light, X-rays or use of unstable cell lines. Different mutagenic agents produce different types of mutations, and the ordinarily skilled artisan will be able to select the appropriate agent to generate the desired type of mutations. For example, ethylmethanesulfonate (EMS) and N-ethyl-N-nitrosourea (ENU) can be used to generate single base pair changes, while X-rays often result in deletions and gross chromosomal rearrangements. UV light exposure produces dimers between adjacent pyrimidines in DNA, which can result in point mutations, deletions and rearrangements.
Error prone cell lines can also be used to introduce mutations, for example on a plasmid comprising a CasX
protein or sgNA scaffold of the disclosure. A population of DNA molecules encoding a CasX
protein (for example, a protein of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3) or an sgNA
scaffold can be exposed to a mutagen to generate collection of CasX variant proteins or sgNA
scaffold variants, and these collections can be assayed for improved function using any of the assays described herein.
j. Staggered Extension Process (StEP)

[00385] In some embodiments, a staggered extension process (StEP) is used to generate CasX
variant proteins or sgNA scaffold variants with improved function. Staggered extension process is a specialized PCR protocol that allows for the breeding of multiple variants of a protein during a PCR reaction. StEP utilizes a polymerase with low processivity, (for example Taq or Vent polymerase) to create short primers off of two or more different template strands with a significant level of sequence similarity. The short primers are then extended for short time intervals allowing for shuffling of the template strands. This method can also be used as a means to stack DME variants. Exemplary StEP protocols are described by Zhao, H. et al. (1998) "Molecular evolution by staggered extension process (StEP) in vitro recombination" Nature Biotechnology 16: 258-261, the contents of which are incorporated herein by reference in their entirety. StEP can be used to generate collections of CasX variant proteins or sgNA scaffold variants, and these collections can be assayed for improved function using any of the assays described herein.
k. Gene Shuffling

[00386] In some embodiments, gene shuffling is used to generate CasX variant proteins or sgNA scaffold variants with improved function. In some embodiments, gene shuffling is used to combine (sometimes referred to herein as "stack") variants produced through other methods described herein, such as plasmid recombineering. In an exemplary gene shuffling protocol, a DNase, for example DNase I, is used to shear a set of parent genes into pieces of 50-100 base pair (bp) in length. In some embodiments, these parent genes comprise CasX
variant proteins with improved function created and isolated using the methods described herein. In some embodiments, these parent genes comprise sgNA scaffold variants with improved function created and isolated using the methods described herein. Dnase fragmentation is then followed by a polymerase chain reaction (PCR) without primers. DNA fragments with sufficient overlapping homologous sequence will anneal to each other and are then extended by DNA
polymerase. If different fragments comprising different mutations anneal, the result is a new variant combining those two mutations. In some embodiments, PCR without primers is followed by PCR extension, and purification of shuffled DNA molecules that have reached the size of the parental genes (e.g., a sequence encoding a CasX protein or sgNA scaffold).
These genes can then be amplified with another PCR, for example by adding PCR primers complementary to the 5' and 3' ends of gene undergoing shuffling. In some embodiments, the primers may have additional sequences added to their 5' ends, such as sequences for restriction enzyme recognition sites needed for ligation into a cloning vector.
1. Domain swapping

[00387] In some embodiments, domain swapping is used to generate CasX variant proteins or sgNA scaffold variants with improved function. To generate CasX variant proteins, engineered domain swapping can be used to mix and match parts with other proteins and CRISPR
molecules. For example, CRISPR proteins have conserved RuvC domains, so the CasX RuvC
domain could be swapped for that of other CRISPR proteins, and the resulting protein assayed for improved DNA cleavage using the assays described herein. For sgNAs, the scaffold stem, extended stem or loops can be exchanged with structures found in other RNAs, for example the scaffold stem and extended stem of the sgNA can be exchanged with thermostable stem loops from other RNAs, and the resulting variant assayed for improved function using the assays described herein. In some embodiments, domain swapping can be used to insert new domains into the CasX protein or sgNA. In some exemplary embodiments where domain swapping is applied to a protein, the inserted domain comprises an entire second protein.
VII. Vectors

[00388] In some embodiments, provided herein are vectors comprising polynucleotides encoding the CasX variant proteins and sgNA or dgNA variants and, optionally, donor template polynucleotides, described herein. In some cases, the vectors are utilized for the expression and recovery of the CasX, gNA (and, optionally, the donor template) components of the gene editing pair. In other cases, the vectors are utilized for the delivery of the encoding polynucleotides to target cells for the editing of the target nucleic acid, as described more fully, below.

[00389] In some embodiments, provided herein are polynucleotides encoding the sgNA or dgNA variants described herein. In some embodiments, said polynucleotides are DNA. In other embodiments, said polynucleotides are RNA. In some embodiments, provided herein are vectors comprising the polynucleotides sequences encoding the sgNA or dgNA
variants described herein. In some embodiments, the vectors comprising the polynucleotides include bacterial plasmids, viral vectors, and the like. In some embodiments, a CasX
variant protein and a sgNA variant are encoded on the same vector. In some embodiments, a CasX
variant protein and a sgNA variant are encoded on different vectors.

[00390] In some embodiments, the disclosure provides a vector comprising a nucleotide sequence encoding the components of the CasX:gNA system. For example, in some embodiments provided herein is a recombinant expression vector comprising a) a nucleotide sequence encoding a CasX variant protein; and b) a nucleotide sequence encoding a gNA variant described herein. In some cases, the nucleotide sequence encoding the CasX
variant protein and/or the nucleotide sequence encoding the gNA variant are operably linked to a promoter that is operable in a cell type of choice (e.g., a prokaryotic cell, a eukaryotic cell, a plant cell, an animal cell, a mammalian cell, a primate cell, a rodent cell, a human cell).
Suitable promoters for inclusion in the vectors are described herein, below.

[00391] In some embodiments, the nucleotide sequence encoding the CasX variant protein is codon optimized. This type of optimization can entail a mutation of a CasX-encoding nucleotide sequence to mimic the codon preferences of the intended host organism or cell while encoding the same protein. Thus, the codons can be changed, but the encoded protein remains unchanged.
For example, if the intended target cell was a human cell, a human codon-optimized CasX
variant-encoding nucleotide sequence could be used. As another non-limiting example, if the intended host cell were a mouse cell, then a mouse codon-optimized CasX
variant-encoding nucleotide sequence could be generated. As another non-limiting example, if the intended host cell were a plant cell, then a plant codon-optimized CasX variant protein-encoding nucleotide sequence could be generated. As another non-limiting example, if the intended host cell were a bacterial cell, then a bacterial codon-optimized CasX variant protein-encoding nucleotide sequence could be generated.

[00392] In some embodiments, provided herein are one or more recombinant expression vectors such as (i) a nucleotide sequence of a donor template nucleic acid wherein the donor template comprises a nucleotide sequence having homology to a target sequence of a target nucleic acid (e.g., a target genome); (ii) a nucleotide sequence that encodes a gNA or a gNA
variant as described herein, that may be provided in a single-guide or dual-guide form, (e.g., operably linked to a promoter that is operable in a target cell such as a eukaryotic cell); and (iii) a nucleotide sequence encoding a CasX protein or a CasX variant protein (e.g., operably linked to a promoter that is operable in a target cell such as a eukaryotic cell). In some embodiments, the sequences encoding the gNA and CasX proteins are in different recombinant expression vectors, and in other embodiments the gNA and CasX proteins are in the same recombinant expression vector. In some embodiments, the sequences encoding the gNA, the CasX protein, and the donor template(s) are in different recombinant expression vectors, and in other embodiments one or more are in the same recombinant expression vector. In some embodiments, either the sgNA in the recombinant expression vector, the CasX
protein encoded by the recombinant expression vector, or both, are variants of a reference CasX protein or gNAs as described herein. In the case of the nucleotide sequence encoding the gNA, the recombinant expression vector can be transcribed in vitro, for example using T7 promoter regulatory sequences and T7 polymerase in order to produce the gRNA, which can then be recovered by conventional methods; e.g., purification via gel electrophoresis. Once synthesized, the gRNA
may be utilized in the gene editing pair to directly contact a target DNA or may be introduced into a cell by any of the well-known techniques for introducing nucleic acids into cells (e.g., microinjection, electroporation, transfection, etc.).

[00393] Depending on the host/vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector.

[00394] In some embodiments, a nucleotide sequence encoding a reference or variant CasX
and/or gNA is operably linked to a control element; e.g., a transcriptional control element, such as a promoter. In some embodiments, a nucleotide sequence encoding a reference or CasX
variant protein is operably linked to a control element; e.g., a transcriptional control element, such as a promoter. In some cases, the promoter is a constitutively active promoter. In some cases, the promoter is a regulatable promoter. In some cases, the promoter is an inducible promoter. In some cases, the promoter is a tissue-specific promoter. In some cases, the promoter is a cell type-specific promoter. In some cases, the transcriptional control element (e.g., the promoter) is functional in a targeted cell type or targeted cell population.
For example, in some cases, the transcriptional control element can be functional in eukaryotic cells, e.g., hematopoietic stem cells (e.g., mobilized peripheral blood (mPB) CD34(+) cell, bone marrow (BM) CD34(+) cell, etc.). By transcriptional activation, it is intended that transcription will be increased above basal levels in the target cell by 10 fold, by 100 fold, more usually by 1000 fold.

[00395] Non-limiting examples of eukaryotic promoters (promoters functional in a eukaryotic cell) include EFlalpha, EFlalpha core promoter, those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, and mouse metallothionein-I. Further non-limiting examples of eukaryotic promoters include the CMV promoter full-length promoter, the minimal CMV
promoter, the chicken 13-actin promoter, the hPGK promoter, the HSV TK
promoter, the Mini-TK promoter, the human synapsin I promoter which confers neuron-specific expression, the Mecp2 promoter for selective expression in neurons, the minimal IL-2 promoter, the Rous sarcoma virus enhancer/promoter (single), the spleen focus-forming virus long terminal repeat (LTR) promoter, the SV40 promoter, the SV40 enhancer and early promoter, the TBG promoter:
promoter from the human thyroxine-binding globulin gene (Liver specific), the PGK promoter, the human ubiquitin C promoter, the UCOE promoter (Promoter of HNRPA2B1-CBX3), the Histone H2 promoter, the Histone H3 promoter, the Ul al small nuclear RNA
promoter (226 nt), the U1b2 small nuclear RNA promoter (246 nt) 26, the TTR minimal enhancer/promoter, the b-kinesin promoter, the human eIF4A1 promoter, the ROSA26 promoter and the Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter.

[00396] Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. The expression vector may also contain a ribosome binding site for translation initiation and a transcription terminator. The expression vector may also include appropriate sequences for amplifying expression. The expression vector may also include nucleotide sequences encoding protein tags (e.g., 6xHis tag, hemagglutinin tag, fluorescent protein, etc.) that can be fused to the CasX protein, thus resulting in a chimeric CasX
polypeptide.

[00397] In some embodiments, a nucleotide sequence encoding a gNA variant and/or a CasX
variant protein is operably linked to a promoter that is an inducible promoter (i.e., a promoter whose state, active/"ON" or inactive/"OFF", is controlled by an external stimulus, e.g., the presence of a particular temperature, compound, or protein) or a promoter that is a constitutively active promoter (i.e., a promoter that is constitutively in an active/"ON"
state). In other embodiments, a nucleotide sequence encoding a gNA variant and/or a CasX
variant protein is operably linked to a spatially restricted promoter (i.e., transcriptional control element, enhancer, tissue specific promoter, cell type specific promoter, etc.), and it may be a temporally restricted promoter (i.e., the promoter is in the "ON" state or "OFF" state during specific stages of embryonic development or during specific stages of a biological process, e.g., hair follicle cycle in mice).

[00398] In certain embodiments, suitable promoters can be derived from viruses and can therefore be referred to as viral promoters, or they can be derived from any organism, including prokaryotic or eukaryotic organisms. Suitable promoters can be used to drive expression by any RNA polymerase (e.g., poll, pol II, pol III). Exemplary promoters include, but are not limited to the 5V40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter;
adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, a human U6 small nuclear promoter (U6), an enhanced U6 promoter, a human HI promoter (HI), a POL1 promoter, a 7SK promoter, tRNA
promoters and the like.

[00399] In some embodiments, a nucleotide sequence encoding a gNA is operably linked to (under the control of) a promoter operable in a eukaryotic cell (e.g., a U6 promoter, an enhanced U6 promoter, an HI promoter, and the like). As would be understood by one of ordinary skill in the art, when expressing an RNA (e.g., a gRNA) from a nucleic acid (e.g., an expression vector) using a U6 promoter (e.g., in a eukaryotic cell), or another PolIII promoter, the RNA may need to be mutated if there are several Ts in a row (coding for Us in the RNA).
This is because a string of Ts (e.g., 5 Ts) in DNA can act as a terminator for polymerase III
(Pol III). Thus, in order to ensure transcription of a gRNA (e.g., the activator portion and/or targeter portion, in dual guide or single guide format) in a eukaryotic cell, it may sometimes be necessary to modify the sequence encoding the gRNA to eliminate runs of Ts. In some cases, a nucleotide sequence encoding a CasX protein (e.g., a wild type CasX protein, a nickase CasX
protein, a dCasX
protein, a chimeric CasX protein and the like) is operably linked to a promoter operable in a eukaryotic cell (e.g., a CMV promoter, an EFlalpha promoter, an estrogen receptor-regulated promoter, and the like).

[00400] In certain embodiments, inducible promoters suitable for use may include any inducible promoter described herein or known to one of ordinary skill in the art. Examples of inducible promoters include, without limitation, T7 RNA polymerase promoter, polymerase promoter, isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoter, lactose induced promoter, chemically/biochemically-regulated and physically-regulated promoters such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline -responsive promoter systems, which include a tetracycline repressor protein (tetR), a tetracycline operator sequence (tet0) and a tetracycline transactivator fusion protein (tTA), steroid-regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid/retinoid/thyroid receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and sequester metal ions) genes from yeast, mouse and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene or benzothiadiazole (BTH)), temperature/heat-inducible promoters (e.g., heat shock promoters), and light-regulated promoters (e.g., light responsive promoters from plant cells).

[00401] In some cases, the promoter is a spatially restricted promoter (i.e., cell type specific promoter, tissue specific promoter, etc.) such that in a multi-cellular organism, the promoter is active (i.e., "ON") in a subset of specific cells. Spatially restricted promoters may also be referred to as enhancers, transcriptional control elements, control sequences, etc. Any convenient spatially restricted promoter may be used as long as the promoter is functional in the targeted host cell (e.g., eukaryotic cell; prokaryotic cell).

[00402] In some cases, the promoter is a reversible promoter. Suitable reversible promoters, including reversible inducible promoters are known in the art. Such reversible promoters may be isolated and derived from many organisms, e.g., eukaryotes and prokaryotes.
Modification of reversible promoters derived from a first organism for use in a second organism, e.g., a first prokaryote and a second a eukaryote, a first eukaryote and a second a prokaryote, etc., is well known in the art. Such reversible promoters, and systems based on such reversible promoters but also comprising additional control proteins, include, but are not limited to, alcohol regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator proteins (AlcR), etc.), tetracycline regulated promoters, (e.g., promoter systems including Tet Activators, TetON, TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, etc.), metal regulated promoters (e.g., metallothionein promoter systems, etc.), pathogenesis-related regulated promoters (e.g., salicylic acid regulated promoters, ethylene regulated promoters, benzothiadiazole regulated promoters, etc.), temperature regulated promoters (e.g., heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light regulated promoters, synthetic inducible promoters, and the like.

[00403] Recombinant expression vectors of the disclosure can also comprise elements that facilitate robust expression of reference or CasX variant proteins and/or reference or variant gNAs of the disclosure. For example, recombinant expression vectors can include one or more of a polyadenylation signal (PolyA), an intronic sequence or a post-transcriptional regulatory element such as a woodchuck hepatitis post-transcriptional regulatory element (WPRE).
Exemplary polyA sequences include hGH poly(A) signal (short), HSV TK poly(A) signal, synthetic polyadenylation signals, 5V40 poly(A) signal, P-globin poly(A) signal and the like. In addition, vectors used for providing a nucleic acid encoding a gNA and/or a CasX protein to a cell may include nucleic acid sequences that encode for selectable markers in the target cells, so as to identify cells that have taken up the gNA and/or CasX protein. A person of ordinary skill in the art will be able to select suitable elements to include in the recombinant expression vectors described herein.

[00404] A recombinant expression vector sequence can be packaged into a virus or virus-like particle (also referred to herein as a "particle" or "virion") for subsequent infection and transformation of a cell, ex vivo, in vitro or in vivo. Such particles or virions will typically include proteins that encapsidate or package the vector genome. In some embodiments, a recombinant expression vector of the present disclosure is a recombinant adeno-associated virus (AAV) vector. In some embodiments, a recombinant expression vector of the present disclosure is a recombinant lentivirus vector. In some embodiments, a recombinant expression vector of the present disclosure is a recombinant retroviral vector.

[00405] Adeno-associated virus (AAV) is a small (20 nm), nonpathogenic virus that is useful in treating human diseases in situations that employ a viral vector for delivery to a cell such as a eukaryotic cell, either in vivo or ex vivo for cells to be prepared for administering to a subject. A
construct is generated, for example a construct encoding any of the CasX
proteins and/or gNA
embodiments as described herein, and is flanked with AAV inverted terminal repeat (ITR) sequences, thereby enabling packaging of the AAV vector into an AAV viral particle.

[00406] An "AAV" vector may refer to the naturally occurring wild-type virus itself or derivatives thereof. The term covers all subtypes, serotypes and pseudotypes, and both naturally occurring and recombinant forms, except where required otherwise. As used herein, the term "serotype" refers to an AAV which is identified by and distinguished from other AAVs based on capsid protein reactivity with defined antisera, e.g., there are many known serotypes of primate AAVs. In some embodiments, the AAV vector is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV-Rh74 (Rhesus macaque-derived AAV), and AAVRh10, and modified capsids of these serotypes. For example, serotype AAV-2 is used to refer to an AAV which contains capsid proteins encoded from the cap gene of AAV-2 and a genome containing 5' and 3' ITR sequences from the same AAV-2 serotype.
Pseudotyped AAV
refers to an AAV that contains capsid proteins from one serotype and a viral genome including 5'-3' ITRs of a second serotype. Pseudotyped rAAV would be expected to have cell surface binding properties of the capsid serotype and genetic properties consistent with the ITR serotype.
Pseudotyped recombinant AAV (rAAV) are produced using standard techniques described in the art. As used herein, for example, rAAV1 may be used to refer an AAV having both capsid proteins and 5'-3' ITRs from the same serotype or it may refer to an AAV
having capsid proteins from serotype 1 and 5'-3' ITRs from a different AAV serotype, e.g., AAV
serotype 2. For each example illustrated herein the description of the vector design and production describes the serotype of the capsid and 5'-3' ITR sequences.

[00407] An "AAV virus" or "AAV viral particle" refers to a viral particle composed of at least one AAV capsid protein (preferably by all of the capsid proteins of a wild-type AAV) and an encapsidated polynucleotide. If the particle additionally comprises a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome to be delivered to a mammalian cell), it is typically referred to as "rAAV". An exemplary heterologous polynucleotide is a polynucleotide comprising a CasX protein and/or sgRNA and, optionally, a donor template of any of the embodiments described herein.

[00408] By "adeno-associated virus inverted terminal repeats" or "AAV ITRs" is meant the art recognized regions found at each end of the AAV genome which function together in cis as origins of DNA replication and as packaging signals for the virus. AAV ITRs, together with the AAV rep coding region, provide for the efficient excision and rescue from, and integration of a nucleotide sequence interposed between two flanking ITRs into a mammalian cell genome. The nucleotide sequences of AAV ITR regions are known. See, for example Kotin, R.M. (1994) Human Gene Therapy 5:793-801; Berns, K. I. "Parvoviridae and their Replication" in Fundamental Virology, 2' Edition, (B. N. Fields and D. M. Knipe, eds.). As used herein, an AAV ITR need not have the wild-type nucleotide sequence depicted, but may be altered, e.g., by the insertion, deletion or substitution of nucleotides. Additionally, the AAV
ITR may be derived from any of several AAV serotypes, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-Rh74, and AAVRhl 0, and modified capsids of these serotypes. Furthermore, 5' and 3' ITRs which flank a selected nucleotide sequence in an AAV vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they function as intended, i.e., to allow for excision and rescue of the sequence of interest from a host cell genome or vector, and to allow integration of the heterologous sequence into the recipient cell genome when AAV Rep gene products are present in the cell. Use of AAV serotypes for integration of heterologous sequences into a host cell is known in the art (see, e.g., W02018195555A1 and U520180258424A1, incorporated by reference herein.).

[00409] By "AAV rep coding region" is meant the region of the AAV genome which encodes the replication proteins Rep 78, Rep 68, Rep 52 and Rep 40. These Rep expression products have been shown to possess many functions, including recognition, binding and nicking of the AAV origin of DNA replication, DNA helicase activity and modulation of transcription from AAV (or other heterologous) promoters. The Rep expression products are collectively required for replicating the AAV genome. By "AAV cap coding region" is meant the region of the AAV
genome which encodes the capsid proteins VP1, VP2, and VP3, or functional homologues thereof These Cap expression products supply the packaging functions which are collectively required for packaging the viral genome.

[00410] In some embodiments, AAV capsids utilized for delivery of the encoding sequences for the CasX and gNA, and, optionally, the donor template nucleotides to a host cell can be derived from any of several AAV serotypes, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-Rh74 (Rhesus macaque-derived AAV), and AAVRh10, and the AAV ITRs are derived from AAV serotype 2.

[00411] In order to produce rAAV viral particles, an AAV expression vector is introduced into a suitable host cell using known techniques, such as by transfection.
Packaging cells are typically used to form virus particles; such cells include HEK293 cells (and other cells known in the art), which package adenovirus. A number of transfection techniques are generally known in the art; see, e.g., Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York. Particularly suitable transfection methods include calcium phosphate co-precipitation, direct microinjection into cultured cells, electroporation, liposome mediated gene transfer, lipid-mediated transduction, and nucleic acid delivery using high-velocity microprojectiles.

[00412] In some embodiments, host cells transfected with the above-described AAV
expression vectors are rendered capable of providing AAV helper functions in order to replicate and encapsidate the nucleotide sequences flanked by the AAV ITRs to produce rAAV viral particles. AAV helper functions are generally AAV-derived coding sequences which can be expressed to provide AAV gene products that, in turn, function in trans for productive AAV
replication. AAV helper functions are used herein to complement necessary AAV
functions that are missing from the AAV expression vectors. Thus, AAV helper functions include one, or both of the major AAV ORFs (open reading frames), encoding the rep and cap coding regions, or functional homologues thereof. Accessory functions can be introduced into and then expressed in host cells using methods known to those of skill in the art. Commonly, accessory functions are provided by infection of the host cells with an unrelated helper virus. In some embodiments, accessory functions are provided using an accessory function vector. Depending on the host/vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc., may be used in the expression vector.

[00413] In other embodiments, retroviruses, for example, lentiviruses, may be suitable for use as vectors for delivery of the encoding nucleic acids of the CasX:gNA systems of the present disclosure. Commonly used retroviral vectors are "defective", e.g. unable to produce viral proteins required for productive infection, and may be referred to a virus-like particles (VLP).
Rather, replication of the vector requires growth in a packaging cell line. To generate viral particles comprising nucleic acids of interest, the retroviral nucleic acids comprising the nucleic acid are packaged into VLP capsids by a packaging cell line. Different packaging cell lines provide a different envelope protein (ecotropic, amphotropic or xenotropic) to be incorporated into the capsid, this envelope protein determining the specificity of the viral particle for the cells (ecotropic for murine and rat; amphotropic for most mammalian cell types including human, dog and mouse; and xenotropic for most mammalian cell types except murine cells).
The appropriate packaging cell line may be used to ensure that the cells are targeted by the packaged viral particles. Methods of introducing subject vector expression vectors into packaging cell lines and of collecting the viral particles that are generated by the packaging lines are well known in the art.

[00414] For non-viral delivery, vectors can also be delivered wherein the vector or vectors encoding the CasX variants and gNA are formulated in nanoparticles, wherein the nanoparticles contemplated include, but are not limited to nanospheres, liposomes, quantum dots, polyethylene glycol particles, hydrogels, and micelles. Lipid nanoparticles are generally composed of an ionizable cationic lipid and three or more additional components, such as cholesterol, DOPE, polylactic acid-co-glycolic acid, and a polyethylene glycol (PEG) containing lipid. In some embodiments, the CasX variants of the embodiments disclosed herein are formulated in a nanoparticle. In some embodiments, the nanoparticle comprises the gNA of the embodiments disclosed herein. In some embodiments, the nanoparticle comprises RNP of the CasX variant complexed with the gNA. In some embodiments, the system comprises a nanoparticle comprising nucleic acids encoding the CasX variants and the gNA and, optionally, a donor template nucleic acid. In some embodiments, the components of the CasX:gNA
system are formulated in separate nanaoparticles for delivery to cells or for administration to a subject in need thereof.

VIII. Applications

[00415] The CasX proteins, guides, nucleic acids, and variants thereof provided herein, as well as vectors encoding such components, are useful for various applications, including therapeutics, diagnostics, and research.

[00416] Provided herein are methods of cleaving a target DNA, comprising contacting the target DNA with a CasX protein and gNA pair. In some embodiments, the pair comprises a CasX variant protein and a gNA, wherein the CasX variant protein is a CasX
variant of SEQ ID
NO: 2 as described herein (e.g., a sequence of Tables 3, 8, 9, 10 and 12), and wherein the contacting results in cleavage and, optionally, editing of the target DNA. In other embodiments, the pair comprises a reference CasX protein and a gNA. In some embodiments, the gNA is a gNA variant of the disclosure (e.g., a sequence of SEQ ID NOS: 2101-2280), or a reference gRNA scaffold comprising SEQ ID NO: 5 or SEQ ID NO: 4, and further comprises a spacer that is complementary to the target DNA.

[00417] In yet further aspects, the disclosure provides methods of cleaving a target DNA, comprising contacting the target DNA with a CasX protein and gNA pair of any of the embodiments described herein, wherein the contacting results in cleavage and optionally editing of the target DNA. In some embodiments, the scaffold of the gNA variant comprises a sequence of SEQ ID NO: 2101-2280, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%
sequence identity thereto, and further comprises a spacer that is complementary to the target DNA. In some embodiments, the CasX protein is a CasX variant protein of any of the embodiments described herein (e.g., a sequence of Tables 3, 8, 9, 10 and 12), or a reference CasX
protein SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[00418] In some embodiments, the methods of editing a target DNA comprise contacting a target DNA with a CasX protein and gNA pair as described herein and a donor polynucleotide, sometimes referred to as a donor template. In some embodiments, CasX protein and gNA pairs generate site-specific double strand breaks (DSBs) or single strand breaks (SSBs) (e.g., when the CasX variant protein is a nickase) within double-stranded DNA (dsDNA) target nucleic acids, which are repaired either by non-homologous end joining (NHEJ), homology-directed repair (HDR), homology-independent targeted integration, micro-homology mediated end joining (MNIEJ), single strand annealing (SSA) or base excision repair (BER). In some cases, contacting a target DNA with a gene editing pair occurs under conditions that are permissive for NHEJ, HDR, or MMEJ. Thus, in some cases, a method as provided herein includes contacting the target DNA with a donor polynucleotide (e.g., by introducing the donor polynucleotide into a cell), wherein the donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of a copy of the donor polynucleotide integrates into the target DNA. For example, an exogenous donor template which may comprise a corrective sequence (or a deletion to knock-out the defective allele) to be integrated flanked by an upstream sequence and a downstream sequence is introduced into a cell. The upstream and downstream sequences relative to the cleavage site(s) share sequence similarity with either side of the site of integration in the target DNA (i.e., homologous arms), facilitating the insertion. In other cases, an exogenous donor template which may comprise a corrective sequence is inserted between the ends generated by CasX cleavage by homology-independent targeted integration (HITT) mechanisms. The exogenous sequence inserted by HITI can be any length, for example, a relatively short sequence of between 1 and 50 nucleotides in length, or a longer sequence of about 50-1000 nucleotides in length. The lack of homology can be, for example, having no more than 20-50% sequence identity and/or lacking in specific hybridization at low stringency.
In other cases, the lack of homology can further include a criterion of having no more than 5, 6, 7, 8, or 9 bp identity. In some cases, the method does not comprise contacting a cell with a donor polynucleotide, and the target DNA is modified such that nucleotides within the target DNA are deleted or inserted according to the cells own repair pathways.

[00419] The donor template sequence may comprise certain sequence differences as compared to the genomic sequence, e.g., restriction sites, nucleotide polymorphisms, selectable markers (e.g., drug resistance genes, fluorescent proteins, enzymes etc.), etc., which may be used to assess for successful insertion of the donor nucleic acid at the cleavage site or in some cases may be used for other purposes (e.g., to signify expression at the targeted genomic locus).
Alternatively, these sequence differences may include flanking recombination sequences such as FLPs, loxP sequences, or the like, that can be activated at a later time for removal of the marker sequence. In some embodiments of the method, the donor polynucleotide comprises at least about 10, at least about 50, at least about 100, or at least about 200, or at least about 300, or at least about 400, or at least about 500, or at least about 600, or at least about 700, or at least about 800, or at least about 900, or at least about 1000, or at least about 10,000, or at least 15,000 nucleotides of a wild-type gene. In other embodiments, the donor polynucleotide comprises at least about 10 to about 15,000 nucleotides, or at least about 200 to about 10,000 nucleotides, or at least about 400 to about 6000 nucleotides, or at least about 600 to about 4000 nucleotides, or at least about 1000 to about 2000 nucleotides of a wild-type gene. In some embodiments, the donor template is a single stranded DNA template or a single stranded RNA
template. In other embodiments, the donor template is a double stranded DNA template.

[00420] In some embodiments, contacting the target DNA with a CasX protein and gNA gene editing pair of the disclosure results in gene editing. In some embodiments, the editing occurs in vitro, outside of a cell, in a cell-free system. In some embodiments, the editing occurs in vitro, inside of a cell, for example in a cell culture system. In some embodiments, the editing occurs in vivo inside of a cell, for example in a cell in an organism. In some embodiments, the cell is a eukaryotic cell. Exemplary eukaryotic cells may include cells selected from the group consisting of a plant cell, a fungal cell, a mammalian cell, a reptile cell, an insect cell, an avian cell, a fish cell, a parasite cell, an arthropod cell, a cell of an invertebrate, a cell of a vertebrate, a rodent cell, a mouse cell, a rat cell, a pig cell, a dog cell, a primate cell, a non-human primate cell, and a human cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is an embryonic stem cell, an induced pluripotent stem cell, a germ cell, a fibroblast, an oligodendrocyte, a glial cell, a hematopoietic stem cell, a neuron progenitor cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, a retinal cell, a cancer cell, a T-cell, a B-cell, an NK cell, a fetal cardiomyocyte, a myofibroblast, a mesenchymal stem cell, an autotransplated expanded cardiomyocyte, an adipocyte, a totipotent cell, a pluripotent cell, a blood stem cell, a myoblast, an adult stem cell, a bone marrow cell, a mesenchymal cell, a parenchymal cell, an epithelial cell, an endothelial cell, a mesothelial cell, fibroblasts, osteoblasts, chondrocytes, exogenous cell, endogenous cell, stem cell, hematopoietic stem cell, bone-marrow derived progenitor cell, myocardial cell, skeletal cell, fetal cell, undifferentiated cell, multi-potent progenitor cell, unipotent progenitor cell, a monocyte, a cardiac myoblast, a skeletal myoblast, a macrophage, a capillary endothelial cell, a xenogenic cell, an allogenic cell, or a post-natal stem cell. In alternative embodiments, the cell is a prokaryotic cell.

[00421] Methods of editing of the disclosure can occur in vitro outside of a cell, in vitro inside of a cell or in vivo inside of a cell. The cell can be in a subject. In some embodiments, editing occurs in the subject having a mutation in an allele of a gene wherein the mutation causes a disease or disorder in the subject. In some embodiments, editing changes the mutation to a wild type allele of the gene. In some embodiments, editing knocks down or knocks out expression of an allele of a gene causing a disease or disorder in the subject. In some embodiments, editing occurs in vitro inside of the cell prior to introducing the cell into a subject. In some embodiments, the cell is autologous or allogeneic.

[00422] Methods of introducing a nucleic acid (e.g., a nucleic acid comprising a donor polynucleotide sequence, one or more nucleic acids encoding a CasX protein and/or a gNA, or variants thereof as described herein) into a cell are known in the art, and any convenient method can be used to introduce a nucleic acid (e.g., an expression construct such as an AAV or virus like particle (VLP; e.g. a capsid derived from one or more components of a retrovirus, described supra) vector comprising the encoded CasX and gNA components, as described, supra) into a cell. Suitable methods include e.g., viral infection, transfection, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, nucleofection, electroporation, direct addition by cell penetrating CasX proteins that are fused to or recruit donor DNA, cell squeezing, calcium phosphate precipitation, direct microinjection, nanoparticle -mediated nucleic acid delivery, and the like.

[00423] Introducing recombinant expression vectors into cells can occur in any suitable culture media and under any suitable culture conditions that promote the survival of the cells.
Introducing recombinant expression vectors into a target cell can be carried out in vivo, in vitro or ex vivo.

[00424] In some embodiments, a CasX variant protein can be provided as RNA.
The RNA can be provided by direct chemical synthesis, or may be transcribed in vitro from a DNA (e.g., a DNA encoding an mRNA comprising a sequence encoding the CasX variant protein).
Once synthesized, the RNA may, for example, be introduced into a cell by any of the well-known techniques for introducing nucleic acids into cells (e.g., microinjection, electroporation, transfection).

[00425] Nucleic acids may be provided to the cells using well-developed transfection techniques, and the commercially available TransMessenger reagents from Qiagen, StemfectTM
RNA Transfection Kit from Stemgent, and TransIT4D-mRNA Transfection Kit from Mirus Bio LLC, Lonza nucleofection, Maxagen electroporation and the like.

[00426] In some embodiments, vectors may be provided directly to a target host cell. For example, cells may be contacted with vectors comprising the subject nucleic acids (e.g., recombinant expression vectors having the donor template sequence and encoding the gNA

variant; recombinant expression vectors encoding the CasX variant protein) such that the vectors are taken up by the cells. Methods for contacting cells with nucleic acid vectors that are plasmids include electroporation, calcium chloride transfection, microinjection, and lipofection are well known in the art. For viral vector delivery, cells can be contacted with viral particles comprising the subject viral expression vectors; e.g., the vectors are viral particles such as AAV
or VLP that comprise polynucleotides that encode the CasX:gNA components or that comprise CasX:gNA RNP. For non-viral delivery, vectors or the CasX:gNA components can also be formulated for delivery in nanoparticles, wherein the nanoparticles contemplated include, but are not limited to nanospheres, liposomes, quantum dots, polyethylene glycol particles, hydrogels, and micelles.

[00427] A nucleic acid comprising a nucleotide sequence encoding a CasX
variant protein is in some cases an RNA. Thus, in some embodiments a CasX variant protein can be introduced into cells as RNA. Methods of introducing RNA into cells are known in the art and may include, for example, direct injection, transfection, or any other method used for the introduction of DNA. A
CasX variant protein may instead be provided to cells as a polypeptide. Such a polypeptide may optionally be fused to a polypeptide domain that increases solubility of the product. The domain may be linked to the polypeptide through a defined protease cleavage site, e.g. a TEV sequence, which is cleaved by TEV protease. The linker may also include one or more flexible sequences, e.g. from 1 to 10 glycine residues. In some embodiments, the cleavage of the fusion protein is performed in a buffer that maintains solubility of the product, e.g. in the presence of from 0.5 to 2 M urea, in the presence of polypeptides and/or polynucleotides that increase solubility, and the like. Domains of interest may include endosomolytic domains, e.g. influenza HA
domain; and other polypeptides that aid in production, e.g. IF2 domain, GST domain, GRPE
domain, and the like. The polypeptide may be formulated for improved stability. For example, the peptides may be PEGylated, where the polyethyleneoxy group provides for enhanced lifetime in the blood stream.

[00428] Additionally or alternatively, a reference or CasX variant protein of the present disclosure may be fused to a polypeptide permeant domain to promote uptake by the cell. A
number of permeant domains are known in the art and may be used in the non-integrating polypeptides of the present disclosure, including peptides, peptidomimetics, and non-peptide carriers. For example, W02017/106569 and U520180363009A1, incorporated by reference herein in its entirety, describe fusion of a Cas protein with one or more nuclear localization sequences (NLS) to facilitate cell uptake. In other embodiments, a permeant peptide may be derived from the third alpha helix of Drosophila melanogaster transcription factor Antennapaedia, referred to as penetratin, which comprises the amino acid sequence RQIKIWFQNRRMKWKK (SEQ ID NO: 398). As another example, the permeant peptide comprises the HIV-1 tat basic region amino acid sequence, which may include, for example, amino acids 49-57 of naturally-occurring tat protein. Other permeant domains include poly-arginine motifs, for example, the region of amino acids 34-56 of HIV-1 rev protein, nona-arginine, octa-arginine, and the like. The site at which the fusion is made may be selected in order to optimize the biological activity, secretion or binding characteristics of the polypeptide.
The optimal site will be determined by routine experimentation.

[00429] A CasX variant protein of the present disclosure may be produced in vitro or by eukaryotic cells or by prokaryotic cells transformed with encoding vectors (described above), and it may be further processed by unfolding, e.g. heat denaturation, dithiothreitol reduction, etc.
and may be further refolded, using methods known in the art. In the case of production of the gNA of the present disclosure, recombinant expression vectors encoding the gNA
can be transcribed in vitro, for example using T7 promoter regulatory sequences and T7 polymerase in order to produce the gRNA, which can then be recovered by conventional methods; e.g., purification via gel electrophoresis. Once synthesized, the gRNA may be utilized in the gene editing pair to directly contact a target DNA or may be introduced into a cell by any of the well-known techniques for introducing nucleic acids into cells (e.g., microinjection, electroporation, transfection, etc.).

[00430] In some embodiments, modifications of interest that do not alter the primary sequence of the CasX variant protein may include chemical derivatization of polypeptides, e.g., acylation, acetylation, carboxylation, amidation, etc. Also included are modifications of glycosylation, e.g.
those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g. by exposing the polypeptide to enzymes which affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences that have phosphorylated amino acid residues, e.g.
phosphotyrosine, phosphoserine, or phosphothreonine.

[00431] In other embodiments, the present disclosure provides nucleic acids encoding a gNA
variant or encoding a CasX variant and reference CasX proteins that have been modified using ordinary molecular biological techniques and synthetic chemistry so as to improve their resistance to proteolytic degradation, to change the target sequence specificity, to optimize solubility properties, to alter protein activity (e.g., transcription modulatory activity, enzymatic activity, etc.) or to render them more suitable. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g. D-amino acids or non-naturally occurring synthetic amino acids. D-amino acids may be substituted for some or all of the amino acid residues.

[00432] A CasX variant protein of the disclosure may be prepared by in vitro synthesis, using conventional methods as known in the art. Various commercial synthetic apparatuses are available, for example, automated synthesizers by Applied Biosystems, Inc., Beckman, etc. By using synthesizers, naturally occurring amino acids may be substituted with unnatural amino acids. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like. If desired, various groups may be introduced into the peptide during synthesis or during expression, which allow for linking to other molecules or to a surface. Thus cysteines can be used to make thioethers, histidines for linking to a metal ion complex, carboxyl groups for forming amides or esters, amino groups for forming amides, and the like.

[00433] A CasX variant protein of the disclosure may also be isolated and purified in accordance with conventional methods of recombinant synthesis. A lysate may be prepared of the expression host and the lysate purified using high performance liquid chromatography (HPLC), exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. For the most part, the compositions which are used will comprise 50% or more by weight of the desired product, more usually 75% or more by weight, preferably 95% or more by weight, and for therapeutic purposes, usually 99.5% or more by weight, in relation to contaminants related to the method of preparation of the product and its purification. Usually, the percentages will be based upon total protein. Thus, in some cases, a CasX
polypeptide, or a CasX fusion polypeptide, of the present disclosure is at least 80% pure, at least 85% pure, at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure (e.g., free of contaminants, non-CasX proteins or other macromolecules, etc.).

[00434] In some embodiments, to induce cleavage or any desired modification to a target nucleic acid (e.g., genomic DNA), or any desired modification to a polypeptide associated with target nucleic acid in an in vitro cell, the gNA variant and/or the CasX
variant protein of the present disclosure and/or the donor template sequence, whether they be introduced as nucleic acids or polypeptides, are provided to the cells for about 30 minutes to about 24 hours, e.g., 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 18 hours, 20 hours, or any other period from about 30 minutes to about 24 hours, which may be repeated with a frequency of about every day to about every 7 days, e.g., every 1.5 days, every 2 days, every 3 days, or any other frequency from about every day to about every 7days. The agent(s) may be provided to the subject cells one or more times, e.g. one time, twice, three times, or more than three times, and the cells allowed to incubate with the agent(s) for some amount of time following each contacting event; e.g., 16-24 hours, after which time the media is replaced with fresh media and the cells are cultured further.

[00435] In some embodiments, the disclosure provides methods of treating a disease in a subject in need thereof comprising modifying a gene in a cell of the subject, the modifying comprising: a) administering to the subject a CasX protein of any of the embodiments described herein and a gNA of any of the embodiments described herein wherein the targeting sequence of the gNA has a sequence that hybridizes with the target nucleic acid; b) a nucleic acid encoding the CasX protein and gNA of any of the embodiments described herein; c) a vector comprising the nucleic acids encoding the CasX and gNA; d) a VLP comprising a CasX:gNA
RNP; or e) combinations thereof. In some embodiments of the method, the CasX protein and the gNA are associated together in a protein complex, for example a ribonuclear protein complex (RNP).

[00436] In other embodiments, the methods of treating a disease in a subject in need thereof comprise administering to the subject a) a CasX protein or a polynucleotide encoding a CasX
protein, b) a guide nucleic acid (gNA) comprising a targeting sequence or a polynucleotide encoding a gNA wherein the targeting sequence of the gNA has a sequence that hybridizes with the target nucleic acid, and c) a donor template comprising at least a portion or the entirety of a gene to be modified.

[00437] In some embodiments of the method of treating a disease, wherein a vector is administered to the subject, the vector is administered at a dose of at least about 1 x 109 vector genomes (vg), at least about 1 x 1010 vg, at least about 1 x 1011 vg, at least about 1 x 1012 vg, at least about 1 x 1013 vg, at least about 1 x 1014 vg, at least about 1 x 1015 vg, or at least about 1 x 1016 vg. The vector can be administered by a route of administration selected from the group consisting of intraparenchymal, intravenous, intra-arterial, intracerebroventricular, intraci sternal, intrathecal, intracranial, intravitreal, subretinal, and intraperitoneal routes.

[00438] A number of therapeutic strategies have been used to design the compositions for use in the methods of treatment of a subject with a disease. In some embodiments, the invention provides a method of treatment of a subject having a disease, the method comprising administering to the subject a CasX:gNA composition or a vector of any of the embodiments disclosed herein according to a treatment regimen comprising one or more consecutive doses using a therapeutically effective dose. In exemplary embodiments the CasX:gNA
composition comprises a CasX variant of any one of SEQ ID NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4412-4415, or a vector encoding the same. In some embodiments of the treatment regimen, the therapeutically effective dose of the composition or vector is administered as a single dose. In other embodiments of the treatment regimen, the therapeutically effective dose is administered to the subject as two or more doses over a period of at least two weeks, or at least one month, or at least two months, or at least three months, or at least four months, or at least five months, or at least six months. In some embodiments of the treatment regiment, the effective doses are administered by a route selected from the group consisting of subcutaneous, intradermal, intraneural, intranodal, intramedullary, intramuscular, intralumbar, intrathecal, subarachnoid, intraventricular, intracapsular, intravenous, intralymphatical, intravitreal, subretinal, or intraperitoneal routes, wherein the administering method is injection, transfusion, or implantation.

[00439] In some embodiments of the methods of treatment of a subject with a disease, the method comprises administering to the subject a CasX:gNA composition as an RNP
within a VLP disclosed herein according to a treatment regimen comprising one or more consecutive doses using a therapeutically effective dose.

[00440] In some embodiments, the administering of the therapeutically effective amount of a CasX:gNA modality, including a vector comprising a polynucleotide encoding a CasX protein and a guide nucleic acid, or the administering of a CasX-gNA composition disclosed herein, to knock down or knock out expression of a gene product to a subject with a disease leads to the prevention or amelioration of the underlying disease such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disease. In some embodiments, the administration of the therapeutically effective amount of the CasX-gNA
modality leads to an improvement in at least one clinically-relevant parameter for a disease.

[00441] In embodiments in which two or more different targeting complexes are provided to the cell (e.g., two gNA comprising two or more different spacers that are complementary to different sequences within the same or different target nucleic acid), the complexes may be provided simultaneously (e.g. as two polypeptides and/or nucleic acids), or delivered simultaneously. Alternatively, they may be provided consecutively, e.g. the targeting complex being provided first, followed by the second targeting complex, etc. or vice versa.

[00442] To improve the delivery of a DNA vector into a target cell, the DNA
can be protected from damage and its entry into the cell facilitated, for example, by using lipoplexes and polyplexes. Thus, in some cases, a nucleic acid of the present disclosure (e.g., a recombinant expression vector of the present disclosure) can be covered with lipids in an organized structure like a micelle, a liposome, or a lipid nanoparticle. When the organized structure is complexed with DNA it is called a lipoplex. There are three types of lipids, anionic (negatively-charged), neutral, or cationic (positively-charged). Lipoplexes that utilize cationic lipids have proven utility for gene transfer. Cationic lipids, due to their positive charge, naturally complex with the negatively charged DNA. Also as a result of their charge, they interact with the cell membrane.
Endocytosis of the lipoplex then occurs, and the DNA is released into the cytoplasm. The cationic lipids also protect against degradation of the DNA by the cell.

[00443] Complexes of polymers with DNA are referred to as polyplexes. Most polyplexes consist of cationic polymers and their production is regulated by ionic interactions. One large difference between the methods of action of polyplexes and lipoplexes is that polyplexes cannot release their DNA load into the cytoplasm, so to this end, co-transfection with endosome -lytic agents (to lyse the endosome that is made during endocytosis) such as inactivated adenovirus must occur. However, this is not always the case; polymers such as polyethylenimine have their own method of endosome disruption as does chitosan and trimethylchitosan.

[00444] Dendrimers, a highly branched macromolecule with a spherical shape, may be also be used to genetically modify stem cells. The surface of the dendrimer particle may be functionalized to alter its properties. In particular, it is possible to construct a cationic dendrimer (i.e., one with a positive surface charge). When in the presence of genetic material such as a DNA plasmid, charge complementarity leads to a temporary association of the nucleic acid with the cationic dendrimer. On reaching its destination, the dendrimer-nucleic acid complex can be taken up into a cell by endocytosis.

[00445] In some cases, a nucleic acid of the disclosure (e.g., an expression vector) includes an insertion site for a guide sequence of interest. For example, a nucleic acid can include an insertion site for a guide sequence of interest, where the insertion site is immediately adjacent to a nucleotide sequence encoding the portion of a gNA variant (e.g. the scaffold region) that does not change when the guide sequence is changed to hybridize to a desired target sequence. Thus, in some cases, an expression vector includes a nucleotide sequence encoding a gNA, except that the portion encoding the spacer sequence portion of the gNA is an insertion sequence (an insertion site). An insertion site is any nucleotide sequence used for the insertion of a spacer in the desired sequence. "Insertion sites" for use with various technologies are known to those of ordinary skill in the art and any convenient insertion site can be used. An insertion site can be for any method for manipulating nucleic acid sequences. For example, in some cases the insertion site is a multiple cloning site (MCS) (e.g., a site including one or more restriction enzyme recognition sequences), a site for ligation independent cloning, a site for recombination based cloning (e.g., recombination based on att sites), a nucleotide sequence recognized by a CRISPR/Cas (e.g. Cas9) based technology, and the like.
IX. Cells

[00446] In still further embodiments, provided herein are cells comprising components of any of the CasX:gNA systems described herein. In some embodiments, the cells comprise any of the gNA variant embodiments as described herein, or the reference gRNA of SEQ ID
NO: 5 or SEQ
ID NO: 4 and further comprises a spacer that is complementary to the target DNA. In some embodiments, the cells further comprise a CasX variant as described herein (e.g, the sequences of Tables 3, 8, 9, 10 and 12 or a reference CasX protein of SEQ ID NO: 1, SEQ
ID NO: 2, or SEQ ID NO. 3). In other embodiments, the cells comprise RNP of any of the CasX:gNA
embodiments described herein. In other embodiments, the disclosure provides cells comprising vectors encoding the CasX:gNA systems of any of the embodiments described herein. In still other embodiments, the cells comprise target DNA that has been edited by the CasX:gNA
embodiments described herein; either to correct a mutation (knock-in) or to knock-down or knock-out a defective gene.

[00447] In some embodiments, the cell is a eukaryotic cell, for example a human cell. In alternative embodiments, the cell is a prokaryotic cell.

[00448] In some embodiments, the cell is a modified cell (e.g., a genetically modified cell) comprising nucleic acid comprising a nucleotide sequence encoding a CasX
variant protein of the disclosure. In some embodiments, the genetically modified cell is genetically modified with an mRNA comprising a nucleotide sequence encoding a CasX variant protein. In some embodiments, the cell is genetically modified with a recombinant expression vector comprising:

a) a nucleotide sequence encoding a CasX variant protein of the present disclosure; and b) a nucleotide sequence encoding a gNA of the disclosure, and, optionally, comprises a nucleotide sequence encoding a donor template. In some cases, such cells are used to produce the individual components or RNP of CasX:gNA systems for use in editing target DNA. In other cases, cells that have been genetically modified in this way may be administered to a subject for purposes such as gene therapy, e.g., to treat a disease or condition caused by a genetic mutation or defect.

[00449] A cell that can serve as a recipient for a CasX variant protein and/or gNA of the present disclosure and/or a nucleic acid comprising a nucleotide sequence encoding a CasX variant protein and/or a gNA variant, can be any of a variety of cells, including, e.g., in vitro cells; in vivo cells; ex vivo cells; primary cells; cells of an immortalized cell line;
cancer cells; animal cells; plant cells; algal cells; fungal cells; etc. A cell can be a recipient of a CasX RNP of the present disclosure. A cell can be a recipient of a single component of a CasX
system of the present disclosure. A cell can be a recipient of a vector encoding the CasX, gNA and, optionally, a donor template of the CasX:gNA systems of any of the embodiments described herein.

[00450] Non-limiting examples of cells that can serve as host cells for production of the CasX:gNA systems disclosed herein include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g., cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, angiosperms, ferns, clubmosses, hornworts, liverworts, mosses, dicotyledons, monocotyledons, etc.), an algal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, and the like), seaweeds (e.g. kelp) a fungal cell (e.g., a yeast cell, a cell from a mushroom), an animal cell, a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal (e.g., an ungulate (e.g., a pig, a cow, a goat, a sheep); a rodent (e.g., a rat, a mouse); a non-human primate; a human; a feline (e.g., a cat); a canine (e.g., a dog);
etc.), and the like. In some cases, the cell is a cell that does not originate from a natural organism (e.g., the cell can be a synthetically made cell; also referred to as an artificial cell).

[00451] In certain embodiments, as provided herein, a cell can be an in vitro cell (e.g., established cultured cell line including, but not limited to HEK293 cells, HEK293T cells, HEK293-F cells, Lenti-X 293T cells, BHK cells, HepG2 cells, Saos-2 cells, HuH7 cells, A549 cells, NSO cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, hybridoma cells, VERO cells, NIH3T3 cells, COS, WI38 cells, MRCS
cells, HeLa, HT1080 cells, or CHO cells). A cell can be an ex vivo cell (cultured cell from an individual). Such cells can be autologous with respect to a subject to be administered said cell(s).
In other embodiments, the cells can be allogeneic with respect to a subject to be administered said cell(s). A cell can be an in vivo cell (e.g., a cell in an individual). A
cell can be an isolated cell. A cell can be a cell inside of an organism. A cell can be an organism. A
cell can be a cell in a cell culture (e.g., in vitro cell culture). A cell can be one of a collection of cells. A cell can be a prokaryotic cell or derived from a prokaryotic cell. A cell can be a bacterial cell or can be derived from a bacterial cell. A cell can be an archaeal cell or derived from an archaeal cell. A
cell can be a eukaryotic cell or derived from a eukaryotic cell. A cell can be a plant cell or derived from a plant cell. A cell can be an animal cell or derived from an animal cell. A cell can be an invertebrate cell or derived from an invertebrate cell. A cell can be a vertebrate cell or derived from a vertebrate cell. A cell can be a mammalian cell or derived from a mammalian cell. A cell can be a rodent cell or derived from a rodent cell. A cell can be a human cell or derived from a human cell. A cell can be a microbe cell or derived from a microbe cell. A cell can be a fungi cell or derived from a fungi cell. A cell can be an insect cell. A cell can be an arthropod cell. A cell can be a protozoan cell. A cell can be a helminth cell.

[00452] Suitable cells may include, in some embodiments, a stem cell (e.g. an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell; a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.); a somatic cell, e.g. a fibroblast, an oligodendrocyte, a glial cell, a hematopoietic stem cell, a neuron progenitor cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, a retinal cell, a cancer cell, a T-cell, a B-cell, a fetal cardiomyocyte, a myofibroblast, a mesenchymal stem cell, an autotransplated expanded cardiomyocyte, an adipocyte, a totipotent cell, a pluripotent cell, a blood stem cell, a myoblast, an adult stem cell, a bone marrow cell, a mesenchymal cell, a parenchymal cell, an epithelial cell, an endothelial cell, a mesothelial cell, fibroblasts, osteoblasts, chondrocytes, exogenous cell, endogenous cell, stem cell, hematopoietic stem cell, bone-marrow derived progenitor cell, myocardial cell, skeletal cell, fetal cell, undifferentiated cell, multi-potent progenitor cell, unipotent progenitor cell, a monocyte, a cardiac myoblast, a skeletal myoblast, a macrophage, a capillary endothelial cell, a xenogenic cell, an allogenic cell, and a post-natal stem cell.

[00453] In some embodiments, the cell is an immune cell. In some cases, the immune cell is a T
cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, or a macrophage. In some cases, the immune cell is a cytotoxic T cell. In some cases, the immune cell is a helper T cell. In some cases, the immune cell is a regulatory T cell (Treg). In some cases, the cell expresses a chimeric antigen receptor.

[00454] In some embodiments, the cell is a stem cell. Stem cells may include, for example, adult stem cells. Adult stem cells can also be referred to as somatic stem cells. In some embodiments, the stem cell is a hematopoietic stem cell (HSC), neural stem cell or a mesenchymal stem cell. In other embodiments, the stem cell is a mesenchymal stem cell (MSC).
MSCs originally derived from the embryonal mesoderm and isolated from adult bone marrow, can differentiate to form muscle, bone, cartilage, fat, marrow stroma, and tendon. Methods of isolating MSC are known in the art; and any known method can be used to obtain MSC.

[00455] A cell in some embodiments is an arthropod cell.
X. Kits and Articles of Manufacture

[00456] In another aspect, provided herein are kits comprising a CasX protein and one or a plurality of gNA of any of the embodiments of the disclosure and a suitable container (for example a tube, vial or plate). In some embodiments, the kit comprises a gNA
variant of the disclosure, or the reference gRNA of SEQ ID NO: 5 or SEQ ID NO: 4. Exemplary gNA variants that can be included comprise a sequence of any one of SEQ ID NO: 2101-2280.

[00457] In some embodiments, the kit comprises a CasX variant protein of the disclosure (e.g. a sequence of Tables 3, 8, 9, 10 and 12), or the reference CasX protein of SEQ
ID NO: 1, SEQ ID
NO: 2, or SEQ ID NO: 3. In exemplary embodiments, a kit of the disclosure comprises a CasX
variant of any one of SEQ ID NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, and 4412-4415. In some embodiments, the kit comprises a CasX variant of any one of SEQ ID
NOS: 247-337, 3498-3501, 3505-3520, 3540-3549 and 4412-4415. In some embodiments, the kit comprises a CasX variant of any one of 3498-3501, 3505-3520, and 3540-3549.

[00458] In some embodiments, the kit comprises a gNA or a vector encoding a gNA, wherein the gNA comprises a sequence selected from the group consisting of SEQ ID NOS:
412-3295. In some embodiments, the gNA comprises a sequence selected from the group consisting of SEQ

ID NOS: 2101-2280. In some embodiments, the gNA comprises a sequence selected from the group consisting of SEQ ID NOS: 2236, 2237, 2238, 2241, 2244, 2248, 2249, and 2259-2280.

[00459] In certain embodiments, provided herein are kits comprising a CasX
protein and gNA
editing pair comprising a CasX variant protein of Tables 3, 8, 9, 10 and 12 and a gNA variant as described herein (e.g., a sequence of Table 2). In exemplary embodiments, a kit of the disclosure comprises a CasX and gNA editing pair, wherein the CasX variant comprises of any one of SEQ
ID NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4412-4415. In some embodiments, the gNA of the gene editing pair comprises any one of SEQ ID NOS:
412-3295.
In some embodiments, the gNA of the gene editing pair comprises any one of SEQ
ID NOS:
2101-2280. In some embodiments, the gNA of the gene editing pair comprises any one of SEQ
ID NOS: 2236, 2237, 2238, 2241, 2244, 2248, 2249, or 2259-2280.

[00460] In some embodiments, the kit further comprises a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing. In some embodiments, the kit further comprises a pharmaceutically acceptable carrier, diluent or excipient.

[00461] In some embodiments, the kit comprises appropriate control compositions for gene editing applications, and instructions for use.

[00462] In some embodiments, the kit comprises a vector comprising a sequence encoding a CasX variant protein of the disclosure, a gNA variant of the disclosure, optionally a donor template, or a combination thereof

[00463] The present description sets forth numerous exemplary configurations, methods, parameters, and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure, but is instead provided as a description of exemplary embodiments. Embodiments of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting embodiments of the disclosure are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered embodiments may be used or combined with any of the preceding or following individually numbered embodiments. This is intended to provide support for all such combinations of embodiments and is not limited to combinations of embodiments explicitly provided below:

Embodiment Set #1:

[00464] Embodiment 1. A variant of a reference CasX protein, wherein the CasX
variant is capable of forming a complex with a guide nucleic acid, and wherein the complex binds a target nucleic acid, and wherein the CasX variant comprises at least one modification in at least one of the following domains of the reference CasX protein:
(a) a non-target strand binding (NTSB) domain that binds to the non-target strand of DNA, wherein the NTSB domain comprises a four-stranded beta sheet;
(b) a target strand loading (TSL) domain that places the target DNA in a cleavage site of the CasX variant, the TSL domain comprising three positively charged amino acids, wherein the three positively charged amino acids bind to the target strand of DNA, (c) a helical I domain that interacts with both the target DNA and a spacer region of a guide RNA, wherein the helical I domain comprises one or more alpha helices;
(d) a helical II domain that interacts with both the target DNA and a scaffold stem of the guide RNA;
(e) an oligonucleotide binding domain (OBD) that binds a triplex region of the guide RNA; and a RuvC DNA cleavage domain;
wherein the CasX variant exhibits at least one improved characteristic as compared to the reference CasX protein.

[00465] Embodiment 2. The CasX variant of Embodiment 1, wherein the reference CasX
comprises the sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or at least 60%
similarity thereto.

[00466] Embodiment 3. The CasX variant of Embodiment 2, wherein the reference CasX
comprises the sequence of SEQ ID NO: 1, or at least 60% similarity thereto.

[00467] Embodiment 4. The CasX variant of Embodiment 2, wherein the reference CasX
comprises the sequence of SEQ ID NO: 2, or at least 60% similarity thereto.

[00468] Embodiment 5. The CasX variant of Embodiment 2, wherein the reference CasX
comprises the sequence of SEQ ID NO: 3, or at least 60% similarity thereto.

[00469] Embodiment 6. The CasX variant of any one of Embodiment 1 to Embodiment 5, wherein the complex binds a target DNA and cleaves the target DNA.

[00470] Embodiment 7. The CasX variant of any one of Embodiment 1 to Embodiment 5, wherein the complex binds a target DNA but does not cleave the target DNA.

[00471] Embodiment 8. The CasX variant of any one of Embodiment 1 to Embodiment 5, wherein the complex binds a target DNA and generates a single stranded nick in the target DNA.

[00472] Embodiment 9. The CasX variant of any one of Embodiment 1 to Embodiment 8, wherein at least one modification comprises at least one amino acid substitution in a domain.

[00473] Embodiment 10. The CasX variant of any one of Embodiment 1 to Embodiment 9, wherein at least one modification comprises at least one amino acid deletion in a domain.

[00474] Embodiment 11. The CasX variant of Embodiment 10, wherein at least one modification comprises the deletion of 1 to 4 consecutive or non-consecutive amino acids in the protein.

[00475] Embodiment 12. The CasX variant of any one of Embodiment 1 to Embodiment 10, wherein modification comprises at least one amino acid insertion in a domain.

[00476] Embodiment 13. The CasX variant of Embodiment 12, wherein at least one modification comprises the insertion of 1 to 4 consecutive or non-consecutive amino acids in a domain.

[00477] Embodiment 14. The CasX variant of any one of 1 to Embodiment 13, having at least 60% similarity to one of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[00478] Embodiment 15. The CasX variant of Embodiment 14, wherein the variant has at least 60% similarity sequence identity to SEQ ID NO: 2.

[00479] Embodiment 16. The CasX variant of any one of Embodiment 1 to Embodiment 15, wherein the improved characteristic is selected from the group consisting of improved folding of the variant, improved binding affinity to the guide RNA, improved binding affinity to the target DNA, altered binding affinity to one or more PAM sequences, improved unwinding of the target DNA, increased activity, improved editing efficiency, improved editing specificity, increased activity of the nuclease, increased target strand loading for double strand cleavage, decreased target strand loading for single strand nicking, decreased off-target cleavage, improved binding of the non-target strand of DNA, improved protein stability, improved protein:guide RNA
complex stability, improved protein solubility, improved protein:guide RNA
complex solubility, improved protein yield, and improved fusion characteristics.

[00480] Embodiment 17. The CasX variant of any one of Embodiment 1 to Embodiment 16, wherein at least one of the at least one improved characteristic of the CasX
variant is at least about 1.1 to about 100,000 times improved relative to the reference protein.

[00481] Embodiment 18. The CasX variant of any one of Embodiment 1 to Embodiment 17, wherein at least one of the at least one improved characteristics of the CasX
variant is at least about 10 to about 100 times improved relative to the reference protein.

[00482] Embodiment 19. The CasX variant any one of Embodiment 1 to Embodiment 18, wherein the CasX variant has about 1.1 to about 100 times increased binding affinity to the guide RNA compared to the protein of SEQ ID NO: 2.

[00483] Embodiment 20. The CasX variant any one of Embodiment 1 to Embodiment 19, wherein the CasX variant has about one to about two times increased binding affinity to the target DNA compared to the protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID
NO: 3.

[00484] Embodiment 21. The CasX variant of any one of Embodiment 1 to Embodiment 20, wherein the CasX protein comprises between 400 and 3000 amino acids.

[00485] Embodiment 22. The CasX variant of any one of Embodiment 1 to Embodiment 21, comprising at least one modification in at least two domains of the reference CasX protein.

[00486] Embodiment 23. The CasX variant of any one of Embodiment 1 to Embodiment 22, comprising two or more modifications in at least one domain of the reference CasX protein.

[00487] Embodiment 24. The CasX variant of any one of Embodiment 1 to Embodiment 23, wherein at least one modification comprises deletion of at least a portion of one domain of the reference CasX protein.

[00488] Embodiment 25. The CasX variant of any one of Embodiment 1 to Embodiment 24, comprising at least one modification of a region of non-contiguous residues that form a channel in which guide RNA:target DNA complexing occurs.

[00489] Embodiment 26. The CasX variant of any one of Embodiment 1 to Embodiment 25, comprising at least one modification of a region of non-contiguous residues that form an interface which binds with the guide RNA.

[00490] Embodiment 27. The CasX variant of any one of Embodiment 1 to Embodiment 26, comprising at least one modification of a region of non-contiguous residues that form a channel which binds with the non-target strand DNA.

[00491] Embodiment 28. The CasX variant of any one of Embodiment 1 to Embodiment 27, comprising at least one modification of a region of non-contiguous residues that form an interface which binds with the PAM.

[00492] Embodiment 29. The CasX variant of any one of Embodiment 1 to Embodiment 28, comprising at least one modification of a region of non-contiguous surface-exposed residues.

[00493] Embodiment 30. The CasX variant of any one of Embodiment 1 to Embodiment 29, comprising at least one modification of a region of non-contiguous residues that form a core through hydrophobic packing in a domain of the variant.

[00494] Embodiment 31. The CasX variant of any one of Embodiment 1 to Embodiment 30, wherein between 2 to 15 residues of the region are charged.

[00495] Embodiment 32. The CasX variant of any one of Embodiment 1 to Embodiment 31, wherein between 2 to 15 residues of the region are polar.

[00496] Embodiment 33. The CasX variant of any one of Embodiment 1 to Embodiment 32, wherein between 2 to 15 residues of the region stack with DNA or RNA bases.

[00497] Embodiment 34. A variant of a reference guide nucleic acid (NA) capable of binding a reference CasX protein, wherein:
the reference nucleic acid comprises a tracrNA sequence and a crNA sequence, wherein:
the tracrNA comprises a scaffold stem loop region comprising an bubble, the tracrNA and the crNA form a stem and a triplex region, and the tracrNA and the crNA are fused, and form a fusion stem loop region;
the variant comprises at least one modification to the reference guide NA, and the variant exhibits at least one improved characteristic compared to the reference guide RNA.

[00498] Embodiment 35. The guide NA variant of Embodiment 34, comprising a tracrRNA
stem loop comprising the sequence ¨UUU-N3-20-UUU¨.

[00499] Embodiment 36. The guide NA variant of Embodiment 34 or Embodiment 35, comprising a crRNA sequence with ¨AAAG¨ in a location 5' to the spacer region.

[00500] Embodiment 37. The guide NA variant of Embodiment 36, wherein the ¨AAAG¨
sequence is immediately 5' to the spacer region.

[00501] Embodiment 38. The guide NA variant of any one of Embodiment 34 to Embodiment 37, wherein the at least one improved characteristic is selected from the group consisting of improved stability, improved solubility, improved resistance to nuclease activity, increased folding rate of the NA, decreased side product formation during folding, increased productive folding, improved binding affinity to a reference CasX protein, improved binding affinity to a target DNA, improved gene editing, and improved specificity.

[00502] Embodiment 39. The guide NA variant of any one of Embodiment 34 to Embodiment 37, wherein at least one modification comprises at least one nucleic acid substitution in a region.

[00503] Embodiment 40. The guide NA variant of any one of Embodiment 34 to Embodiment 39, wherein at least one modification comprises at least one nucleic acid deletion in a region.

[00504] Embodiment 41. The guide NA variant of Embodiment 40, wherein at least one modification comprises deletion of 1 to 4 nucleic acids in a region.

[00505] Embodiment 42. The guide NA variant of any one of Embodiment 34 to Embodiment 40, wherein at least one modification comprises at least one nucleic acid insertion in a region.

[00506] Embodiment 43. The guide NA variant of Embodiment 42, wherein at least one modification comprises insertion of 1 to 4 nucleic acids in a region.

[00507] Embodiment 44. The guide NA variant of any one of Embodiment 34 to Embodiment 42, comprising a scaffold region at least 60% homologous to SEQ ID NO: 5.

[00508] Embodiment 45. The guide NA variant of any one of Embodiment 34 to Embodiment 44, comprising a scaffold NA stem loop at least 60% homologous to SEQ ID NO:
6.

[00509] Embodiment 46. The guide NA variant of any one of Embodiment 34 to Embodiment 45, comprising an extended stem loop at least 60% homologous to SEQ ID NO: 7.

[00510] Embodiment 47. The guide NA variant of any one of Embodiment 34 to Embodiment 46, wherein the guide NA variant sequence is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% homologous to SEQ ID NO: 4.

[00511] Embodiment 48. The guide NA variant of any one of Embodiment 34 to Embodiment 47, comprising an extended stem loop region comprising fewer than 10,000 nucleotides.

[00512] Embodiment 49. The guide NA variant of any one of Embodiment 34 to Embodiment 44, wherein the scaffold stem loop or the extended stem loop is swapped for an exogenous stem loop.

[00513] Embodiment 50. The guide NA variant of any one of Embodiment 34 to Embodiment 49, further comprising a hairpin loop that is capable of binding a protein, RNA or DNA.

[00514] Embodiment 51. The guide NA variant of Embodiment 50, wherein the hairpin loop is from M52, QB, U1A, or PP7.

[00515] Embodiment 52. The guide NA variant of any one of Embodiment 34 to Embodiment 48, further comprising one or more ribozymes.

[00516] Embodiment 53. The guide NA variant of Embodiment 52, wherein the one or more ribozymes are independently fused to a terminus of the guide RNA variant.

[00517] Embodiment 54. The guide NA variant of Embodiment 52 or Embodiment 53, wherein at least one of the one or more ribozymes are an hepatitis delta virus (HDV) ribozyme, hammerhead ribozyme, pistol ribozyme, hatchet ribozyme, or tobacco ringspot virus (TRSV) ribozyme.

[00518] Embodiment 55. The guide NA variant of any one of Embodiment 34 to Embodiment 54, further comprising a protein binding motif

[00519] Embodiment 56. The guide NA variant of any one of Embodiment 34 to Embodiment 55, further comprising a thermostable stem loop.

[00520] Embodiment 57. The guide NA variant of Embodiment 34, comprising the sequence of any one of SEQ ID NO: 9 to SEQ ID NO: 66.

[00521] Embodiment 58. The guide NA variant of any one of Embodiment 34 to Embodiment 57, further comprising a spacer region.

[00522] Embodiment 59. The guide NA variant of any one of Embodiment 34 to Embodiment 58, wherein the reference guide RNA comprises SEQ ID NO: 5.

[00523] Embodiment 60. The guide NA variant of any one of Embodiment 38 to Embodiment 59, wherein the reference CasX protein comprises SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID
NO: 3.

[00524] Embodiment 61. A gene editing pair comprising a CRISPR-associated protein (Cas protein) and a guide NA, wherein the Cas protein is a CasX variant of any one of Embodiment 1 to Embodiment 33.

[00525] Embodiment 62. The gene editing pair of 61, wherein the guide NA is a guide NA
variant of any one of Embodiment 34 to Embodiment 60, or the guide NA of SEQ
ID NO: 4 or SEQ ID NO: 5.

[00526] Embodiment 63. The gene editing pair of Embodiment 61 or Embodiment 62, wherein the gene editing pair has one or more improved characteristics compared to a gene editing pair comprising a CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; and a guide RNA of SEQ ID NO: 4 or SEQ ID NO: 5.

[00527] Embodiment 64. The gene editing pair of Embodiment 63, wherein the one or more improved characteristics comprises improved protein:guide NA complex stability, improved protein:guide NA complex stability, improved binding affinity between the protein and guide NA, improved kinetics of complex formation, improved binding affinity to the target DNA, improved unwinding of the target DNA, increased activity, improved editing efficiency, improved editing specificity, increased activity of the nuclease, increased target strand loading for double strand cleavage, decreased target strand loading for single strand nicking, decreased off-target cleavage, improved binding of the non-target strand of DNA, or improved resistance to nuclease activity.

[00528] Embodiment 65. A gene editing pair comprising a CRISPR-associated protein (Cas protein) and a guide NA, wherein the guide NA is a guide NA variant of any one of Embodiment 34 to Embodiment 60.

[00529] Embodiment 66. The gene editing pair of Embodiment 65, wherein the Cas protein is a CasX variant of any one of Embodiment 1 to Embodiment 22, or a CasX protein of SEQ ID NO:
1, SEQ ID NO: 2, or SEQ ID NO. 3.

[00530] Embodiment 67. The gene editing pair of Embodiment 65 or Embodiment 66, wherein the gene editing pair has one or more improved characteristics.

[00531] Embodiment 68. The gene editing pair of Embodiment 67, wherein the one or more improved characteristics comprises improved protein:guide NA complex stability, improved protein:guide NA complex stability, improved binding affinity between the protein and guide NA, improved binding affinity to the target DNA, improved unwinding of the target DNA, increased activity, improved editing efficiency, improved editing specificity, increased activity of the nuclease, increased target strand loading for double strand cleavage, decreased target strand loading for single strand nicking, decreased off-target cleavage, improved binding of the non-target strand of DNA, or improved resistance to nuclease activity.

[00532] Embodiment 69. A method of editing a target DNA, comprising combining the target DNA with a gene editing pair, the gene editing pair comprising a CasX variant and a guide RNA, wherein the CasX variant is a CasX variant of any one of Embodiment 1 to Embodiment 33, and wherein the combining results in editing of the target DNA.

[00533] Embodiment 70. The method of 69, wherein the guide NA is a guide NA
variant of any one of Embodiment 34 to Embodiment 60, or the guide RNA of SEQ ID NO: 4 or SEQ ID
NO: 5.

[00534] Embodiment 71. The method of Embodiment 69 or Embodiment 70, wherein editing occurs in vitro outside of a cell.

[00535] Embodiment 72. The method of Embodiment 69 or Embodiment 70, wherein editing occurs in vitro inside of a cell.

[00536] Embodiment 73. The method of Embodiment 69 or Embodiment 70, wherein editing occurs in vivo inside of a cell.

[00537] Embodiment 74. The method of any one of Embodiment 71 to Embodiment 73, wherein the cell is a eukaryotic cell.

[00538] Embodiment 75. The method of Embodiment 74, wherein the eukaryotic cell is selected from the group consisting of a plant cell, a fungal cell, a protist cell, a mammalian cell, a reptile cell, an insect cell, an avian cell, a fish cell, a parasite cell, an arthropod cell, a cell of an invertebrate, a cell of a vertebrate, a rodent cell, a mouse cell, a rat cell, a primate cell, a non-human primate cell, and a human cell.

[00539] Embodiment 76. The method of any one of Embodiment 71 to Embodiment 73, wherein the cell is a prokaryotic cell.

[00540] Embodiment 77. A method of editing a target DNA, comprising combining the target DNA with a gene editing pair, the gene editing pair comprising a CRISPR-associated protein (Cas protein) and a guide NA variant, wherein the guide NA variant is a guide NA variant of any one of Embodiment 34 to Embodiment 60, and wherein the combining results in editing of the target DNA.

[00541] Embodiment 78. The method of Embodiment 77, wherein the Cas protein is a CasX
variant of any one of Embodiment 1 to Embodiment 33, or a CasX protein of SEQ
ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[00542] Embodiment 79. The method of Embodiment 77 or Embodiment 78, wherein editing occurs in vitro outside of a cell.

[00543] Embodiment 80. The method of Embodiment 77 or Embodiment 78, wherein editing occurs in vitro inside of a cell.

[00544] Embodiment 81. The method of Embodiment 77 or Embodiment 78, wherein contacting occurs in vivo inside of a cell.

[00545] Embodiment 82. The method of any one of Embodiment 79 to Embodiment 81, wherein the cell is a eukaryotic cell.

[00546] Embodiment 83. The method of Embodiment 82, wherein the eukaryotic cell is selected from the group consisting of a plant cell, a fungal cell, a mammalian cell, a reptile cell, an insect cell, an avian cell, a fish cell, a parasite cell, an arthropod cell, a cell of an invertebrate, a cell of a vertebrate, a rodent cell, a mouse cell, a rat cell, a primate cell, a non-human primate cell, and a human cell.

[00547] Embodiment 84. The method of any one of Embodiment 79 to Embodiment 81, wherein the cell is a prokaryotic cell.

[00548] Embodiment 85. A cell comprising a CasX variant, wherein the CasX
variant is a CasX variant of any one of Embodiment lto Embodiment33.

[00549] Embodiment 86. The cell of Embodiment 85, further comprising a guide NA variant of any one of Embodiment 34to Embodiment 60, or the guide RNA of SEQ ID NO: 4 or SEQ ID
NO: 5.

[00550] Embodiment 87. A cell comprising a guide NA variant, wherein the guide NA variant is a guide NA variant of any one of Embodiment 34to Embodiment 60.

[00551] Embodiment 88. The cell of Embodiment 87, further comprising a CasX
variant of any one of Embodiment lto Embodiment 33, or a CasX protein of SEQ ID NO: 1, SEQ ID NO:
2, or SEQ ID NO. 3.

[00552] Embodiment 89. The cell of any one of 85to Embodiment 88, wherein the cell is a eukaryotic cell.

[00553] Embodiment 90. The cell of any one of 85to Embodiment 88, wherein the cell is a prokaryotic cell.

[00554] Embodiment 91. A polynucleotide encoding the CasX variant of any one of Embodiment lto Embodiment 33.

[00555] Embodiment 92. A vector comprising the polynucleotide of Embodiment 91.

[00556] Embodiment 93. The vector of Embodiment 92, wherein the vector is a bacterial plasmid.

[00557] Embodiment 94. A cell comprising the polynucleotide of Embodiment 91, or the vector of Embodiment 92 or Embodiment 93.

[00558] Embodiment 95. A composition, comprising the CasX variant of any one of Embodiment lto Embodiment 33.

[00559] Embodiment 96. The composition of 95, further comprising a guide RNA
variant of any one of Embodiment 34 to Embodiment 60, or the guide RNA of SEQ ID NO: 4 or SEQ ID
NO: 5.

[00560] Embodiment 97. The composition of Embodiment 95 or Embodiment 96, further comprising a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

[00561] Embodiment 98. A composition, comprising a guide RNA variant of any one of Embodiment 34 to Embodiment 60.

[00562] Embodiment 99. The composition of Embodiment 98, further comprising the CasX
variant of any one of 1 to Embodiment 33, or the CasX protein of SEQ ID NO: 1, SEQ ID NO:
2, or SEQ ID NO: 3.

[00563] Embodiment 100. The composition of Embodiment 98 or Embodiment 99, further comprising a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

[00564] Embodiment 101. A composition, comprising the gene editing pair of any one of Embodiment 61to Embodiment 68.

[00565] Embodiment 102. The composition of Embodiment 101, further comprising a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

[00566] Embodiment 103. A kit, comprising the CasX variant of any one of Embodiment lto Embodiment 33 and a container.

[00567] Embodiment 104. The kit of Embodiment 103, further comprising a guide NA variant of any one of Embodiment 34to Embodiment 60, or the guide RNA of SEQ ID NO: 4 or SEQ ID
NO: 5.

[00568] Embodiment 105. The kit of Embodiment 103 or Embodiment 104, further comprising a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

[00569] Embodiment 106. A kit, comprising a guide NA variant of any one of Embodiment 34to Embodiment 60.

[00570] Embodiment 107. The kit of 106, further comprising the CasX variant of any one of Embodiment 1 to Embodiment 33, or the CasX protein of SEQ ID NO: 1, SEQ ID NO:
2, or SEQ ID NO: 3.

[00571] Embodiment 108. The kit of Embodiment 106 or Embodiment 107, further comprising a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

[00572] Embodiment 109. A kit, comprising the gene editing pair of any one of Embodiment 61 to Embodiment 68.

[00573] Embodiment 110. The kit of Embodiment 109, further comprising a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

[00574] Embodiment 111. A CasX variant comprising any one of the sequences listed in Table 3.

[00575] Embodiment 112. A guide RNA variant comprising any one of the sequences listed in Table 1 or Table 2.

[00576] Embodiment 113. The CasX variant of any one of Embodiment 1 to Embodiment 33, wherein the reference CasX protein comprises a first domain from a first CasX
protein and second domain from a second CasX protein.

[00577] Embodiment 114. The CasX variant of Embodiment 113, wherein the first domain is selected from the group consisting of the NTSB, TSL, helical I, helical II, OBD, and RuvC
domains.

[00578] Embodiment 115. The CasX variant of Embodiment 113, wherein the second domain is selected from the group consisting of the NTSB, TSL, helical I, helical II, OBD, and RuvC
domains.

[00579] Embodiment 116. The method of any one of Embodiment 113 to Embodiment 115, wherein the first and second domains are not the same domain.

[00580] Embodiment 117. The CasX variant of any one of Embodiment 113 to Embodiment 116, wherein the first CasX protein comprises a sequence of SEQ ID NO: 1 and the second CasX protein comprises a sequence of SEQ ID NO: 2.

[00581] Embodiment 118. The CasX variant of any one of Embodiment 113 to Embodiment 116, wherein the first CasX protein comprises a sequence of SEQ ID NO: 1 and the second CasX protein comprises a sequence of SEQ ID NO: 3.

[00582] Embodiment 119. The CasX variant of any one of Embodiment 113 to Embodiment 116, wherein the first CasX protein comprises a sequence of SEQ ID NO: 2 and the second CasX protein comprises a sequence of SEQ ID NO: 3.

[00583] Embodiment 120. The CasX variant of any one of Embodiment 1 to Embodiment 33 or 113to Embodiment 119, wherein the CasX protein comprises at least one chimeric domain comprising a first part from a first CasX protein and a second part from a second CasX protein.

[00584] Embodiment 121. The CasX variant of Embodiment 120, wherein the at least one chimeric domain is selected from the group consisting of the NTSB, TSL, helical I, helical II, OBD, and RuvC domains.

[00585] Embodiment 122. The CasX variant of Embodiment 120 or Embodiment 121, wherein the first CasX protein comprises a sequence of SEQ ID NO: 1 and the second CasX protein comprises a sequence of SEQ ID NO: 2.

[00586] Embodiment 123. The CasX variant of Embodiment 120 or Embodiment 121, wherein the first CasX protein comprises a sequence of SEQ ID NO: 1 and the second CasX protein comprises a sequence of SEQ ID NO: 3.

[00587] Embodiment 124. The CasX variant of Embodiment 120 or Embodiment 121, wherein the first CasX protein comprises a sequence of SEQ ID NO: 2 and the second CasX protein comprises a sequence of SEQ ID NO: 3.

[00588] Embodiment 125. The CasX variant of Embodiment 120, wherein the at least one chimeric comprises a chimeric RuvC domain.

[00589] Embodiment 126. The CasX variant of 125, wherein the chimeric RuvC
domain comprises amino acids 661to Embodiment 824 of SEQ ID NO: 1 and amino acids 922to Embodiment 978 of SEQ ID NO: 2.

[00590] Embodiment 127. The CasX variant of 125, wherein the chimeric RuvC
domain comprises amino acids 648 to 812 of SEQ ID NO: 2 and amino acids 935 to 986 of SEQ ID NO:
1.

[00591] Embodiment 128. The guide NA variant of any one of 34 to Embodiment 60, wherein the reference guide NA comprises a first region from a first guide NA and a second region from a second guide NA.

[00592] Embodiment 129. The guide NA variant of 128, wherein the first region is selected from the group consisting of a triplex region, a scaffold stem loop, and an extended stem loop.

[00593] Embodiment 130. The guide NA variant of 128 or 129, wherein the second region is selected from the group consisting of a triplex region, a scaffold stem loop, and an extended stem loop.

[00594] Embodiment 131. The guide NA variant of any one of Embodiments 128 to Embodiment 130, wherein the first and second regions are not the same region.

[00595] Embodiment 132. The guide NA variant of any one of Embodiments 128 to Embodiment 131, wherein the first guide NA comprises a sequence of SEQ ID NO:
4 and the second guide NA comprises a sequence of SEQ ID NO: 5.

[00596] Embodiment 133. The guide NA variant of any one of Embodiments 34-60 or Embodiments 128-132, comprising at least one chimeric region comprising a first part from a first guide NA and a second part from a second guide NA.

[00597] Embodiment 134. The guide NA variant of Embodiment 133, wherein the at least one chimeric region is selected from the group consisting of a triplex region, a scaffold stem loop, and an extended stem loop.

[00598] Embodiment 135. The guide NA variant of Embodiment 134, wherein the first guide NA comprises a sequence of SEQ ID NO: 4 and the second guide NA comprises a sequence of SEQ ID NO: 5.
Embodiment Set #2

[00599] Embodiment 1. A variant of a reference CasX protein, wherein the CasX
variant is capable of forming a complex with a guide nucleic acid (gNA), and wherein the complex can bind a target nucleic acid, and wherein the CasX variant comprises at least one modification in at least one domain of the reference CasX protein selected from:
a. a non-target strand binding (NTSB) domain that binds to the non-target strand of DNA, wherein the NTSB domain comprises a four-stranded beta sheet;
b. a target strand loading (TSL) domain that places the target DNA in a cleavage site of the CasX variant, the TSL domain comprising three positively charged amino acids, wherein the three positively charged amino acids bind to the target strand of DNA, c. a helical I domain that interacts with both the target DNA and a targeting sequence of a gNA, wherein the helical I domain comprises one or more alpha helices;
d. a helical II domain that interacts with both the target DNA and a scaffold stem of the gNA;
e. an oligonucleotide binding domain (OBD) that binds a triplex region of the gNA; or f. a RuvC DNA cleavage domain;
wherein the CasX variant exhibits one or more improved characteristics as compared to the reference CasX protein.

[00600] Embodiment 2. The CasX variant of Embodiment 1, wherein the CasX
reference comprises the sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[00601] Embodiment 3. The CasX variant of Embodiment 1 or Embodiment 2, wherein the at least one modification comprises at least one amino acid substitution in a domain of the CasX
variant.

[00602] Embodiment 4. The CasX variant of any one of the preceding Embodiments, wherein the at least one modification comprises the substitution of 1 to 10 consecutive or non-consecutive amino acid substitutions in the CasX variant.

[00603] Embodiment 5. The CasX variant of any one of the preceding Embodiments, wherein at least one modification comprises at least one amino acid deletion in a domain of the CasX
variant.

[00604] Embodiment 6. The CasX variant of any one of the preceding Embodiments, wherein the at least one modification comprises the deletion of 1 to 10 consecutive or non-consecutive amino acids in the CasX variant.

[00605] Embodiment 7. The CasX variant of any one of the preceding Embodiments, wherein the at least one modification comprises the substitution of 1 to 10 consecutive or non-consecutive amino acid substitutions and the deletion of 1 to 10 consecutive or non-consecutive amino acids in the CasX variant.

[00606] Embodiment 8. The CasX variant of any one of the preceding Embodiments, wherein the at least one modification comprises at least one amino acid insertion in a domain of the CasX
variant.

[00607] Embodiment 9. The CasX variant of any one of the preceding Embodiments, wherein the at least one modification comprises the insertion of 1 to 4 consecutive or non-consecutive amino acids in a domain of the CasX variant.

[00608] Embodiment 10. The CasX variant of any one of the preceding Embodiments, wherein the CasX variant has a sequence selected from the group consisting of the sequences of Table 3, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, sequence identity thereto.

[00609] Embodiment 11. The CasX variant of any one of the preceding Embodiments, wherein the CasX protein has binding affinity for a protospacer adjacent motif (PAM) sequence selected from the group consisting of TTC, ATC, GTC, and CTC.

[00610] Embodiment 12. The CasX variant of any one of the preceding Embodiments, wherein the CasX protein further comprises one or more nuclear localization signals (NLS).

[00611] Embodiment 13. The CasX variant of Embodiment 12, wherein the one or more NLS
are selected from the group of sequences consisting of PKKKRKV (SEQ ID NO:
352), KRPAATKKAGQAKKKK (SEQ ID NO: 353), PAAKRVKLD (SEQ ID NO: 354), RQRRNELKRSP (SEQ ID NO: 355), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 356), RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 357), VSRKRPRP (SEQ ID NO: 358), PPKKARED (SEQ ID NO: 359), PQPKKKPL (SEQ ID NO:
360), SALIKKKKKMAP (SEQ ID NO: 361), DRLRR (SEQ ID NO: 362), PKQKKRK (SEQ
ID NO: 363), RKLKKKIKKL (SEQ ID NO: 364), REKKKFLKRR (SEQ ID NO: 365), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 366), RKCLQAGMNLEARKTKK (SEQ ID
NO: 367), PRPRKIPR (SEQ ID NO: 368), PPRKKRTVV (SEQ ID NO: 369), NLSKKKKRKREK (SEQ ID NO: 370), RRPSRPFRKP (SEQ ID NO: 371), KRPRSPSS (SEQ
ID NO: 372), KRGINDRNFWRGENERKTR (SEQ ID NO: 373), PRPPKMARYDN (SEQ ID
NO: 374), KRSFSKAF (SEQ ID NO: 375), KLKIKRPVK (SEQ ID NO: 376), PKTRRRPRRSQRKRPPT (SEQ ID NO: 378), RRKKRRPRRKKRR (SEQ ID NO: 381), PKKKSRKPKKKSRK (SEQ ID NO: 382), HKKKHPDASVNFSEFSK (SEQ ID NO: 383), QRPGPYDRPQRPGPYDRP (SEQ ID NO: 384), LSPSLSPLLSPSLSPL (SEQ ID NO: 385), RGKGGKGLGKGGAKRHRK (SEQ ID NO: 386), PKRGRGRPKRGRGR (SEQ ID NO: 387), and MSRRRKANPTKLSENAKKLAKEVEN (SEQ ID NO: 411).

[00612] Embodiment 14. The CasX variant of Embodiment 12 or Embodiment 13, wherein the one or more NLS are expressed at the C-terminus of the CasX protein.

[00613] Embodiment 15. The CasX variant of Embodiment 12 or Embodiment 13, wherein the one or more NLS are expressed at the N-terminus of the CasX protein.

[00614] Embodiment 16. The CasX variant of Embodiment 12 or Embodiment 13, wherein the one or more NLS are expressed at the N-terminus and C-terminus of the CasX
protein.

[00615] Embodiment 17. The CasX variant of any one of the preceding Embodiments, wherein the improved characteristic is selected from the group consisting of improved folding of the variant, improved binding affinity to the gNA, improved binding affinity to the target DNA, altered binding affinity to one or more PAM sequences of the target DNA, improved unwinding of the target DNA, increased activity, improved editing efficiency, improved editing specificity, increased activity of the nuclease, increased target strand loading for double strand cleavage, decreased target strand loading for single strand nicking, decreased off-target cleavage, improved binding of the non-target DNA strand, improved protein stability, improved protein:gNA complex stability, improved protein solubility, improved protein:gNA complex solubility, improved protein yield, improved protein expression, and improved fusion characteristics.

[00616] Embodiment 18. The CasX variant of any one of the preceding Embodiments, wherein at least one or more of the improved characteristics of the CasX variant is at least about 1.1 to about 100,000-fold improved relative to the reference CasX protein of SEQ ID
NO: 1, SEQ ID
NO: 2, or SEQ ID NO: 3.

[00617] Embodiment 19. The CasX variant of any one of the preceding Embodiments, wherein one or more of the improved characteristics of the CasX variant is at least about 10 to about 100-fold improved relative to the reference CasX protein of SEQ ID NO: 1, SEQ
ID NO: 2, or SEQ ID NO: 3.

[00618] Embodiment 20. The CasX variant any one of the preceding Embodiments, wherein the CasX variant has about 1.1 to about 100-fold increased binding affinity to the gNA compared to the protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[00619] Embodiment 21. The CasX variant any one of the preceding Embodiments, wherein the CasX variant has about 1.1 to about 10-fold increased binding affinity to the target DNA
compared to the protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[00620] Embodiment 22. The CasX variant of any one of the preceding Embodiments, wherein the CasX variant comprises between 400 and 3000 amino acids.

[00621] Embodiment 23. The CasX variant of any one of the preceding Embodiments, comprising at least one modification in at least two domains of the CasX
variant relative to the reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[00622] Embodiment 24. The CasX variant of any one of the preceding Embodiments, comprising two or more modifications in at least one domain of the CasX
variant relative to the reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[00623] Embodiment 25. The CasX variant of any one of the preceding Embodiments, wherein at least one modification comprises deletion of at least a portion of one domain of the CasX
variant relative to the reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:
3.

[00624] Embodiment 26. The CasX variant of any one of the preceding Embodiments, comprising at least one modification of a region of non-contiguous amino acid residues of the CasX variant that form a channel in which gNA:target DNA complexing with the CasX variant occurs.

[00625] Embodiment 27. The CasX variant of any one of the preceding Embodiments, comprising at least one modification of a region of non-contiguous amino acid residues of the CasX variant that form an interface which binds with the gNA.

[00626] Embodiment 28. The CasX variant of any one of the preceding Embodiments, comprising at least one modification of a region of non-contiguous amino acid residues of the CasX variant that form a channel which binds with the non-target strand DNA.

[00627] Embodiment 29. The CasX variant of any one of the preceding Embodiments, comprising at least one modification of a region of non-contiguous amino acid residues of the CasX variant that form an interface which binds with the PAM.

[00628] Embodiment 30. The CasX variant of any one of the preceding Embodiments, comprising at least one modification of a region of non-contiguous surface-exposed amino acid residues of the CasX variant.

[00629] Embodiment 31. The CasX variant of any one of the preceding Embodiments, comprising at least one modification of a region of non-contiguous amino acid residues that form a core through hydrophobic packing in a domain of the CasX variant.

[00630] Embodiment 32. The CasX variant of any one of Embodiments 25-30, wherein the modification is a deletion, an insertion, and/or a substitution of one or more amino acids of the region.

[00631] Embodiment 33. The CasX variant of any one of Embodiments 25- 32, wherein between 2 to 15 amino acid residues of the region of the CasX variant are substituted with charged amino acids.

[00632] Embodiment 34. The CasX variant of any one of Embodiments 25- 32, wherein between 2 to 15 amino acid residues of a region of the CasX variant are substituted with polar amino acids.

[00633] Embodiment 35. The CasX variant of any one of Embodiments 25- 32, wherein between 2 to 15 amino acid residues of a region of the CasX variant are substituted with amino acids that stack with DNA or RNA bases.

[00634] Embodiment 36. The CasX variant of any one of the preceding Embodiments, wherein the CasX variant protein comprises a nuclease domain having nickase activity.

[00635] Embodiment 37. The CasX variant of any one of Embodiments 1-35, wherein the CasX variant protein comprises a nuclease domain having double-stranded cleavage activity.

[00636] Embodiment 38. The CasX variant of any one of Embodiments 1-35, wherein the CasX protein is a catalytically inactive CasX (dCasX) protein, and wherein the dCasX and the gNA retain the ability to bind to the target nucleic acid.

[00637] Embodiment 39. The CasX variant of Embodiment 38, wherein the dCasX
comprises a mutation at residues:
a. D672, E769, and/or D935 corresponding to the CasX protein of SEQ ID NO:1;
or b. D659, E756 and/or D922 corresponding to the CasX protein of SEQ ID NO: 2.

[00638] Embodiment 40. The CasX variant of Embodiment 39, wherein the mutation is a substitution of alanine for the residue.

[00639] Embodiment 41. A variant of a reference guide nucleic acid (gNA) capable of binding a CasX protein, wherein the reference guide nucleic acid comprises a tracrNA
sequence and a crNA sequence, wherein:
a. the tracrNA comprises a scaffold stem loop region comprising a bubble;
b. the tracrNA and the crNA form a stem and a triplex region; and c. the tracrNA and the crNA are fused, and form a fusion stem loop region wherein the gNA variant comprises at least one modification compared to the reference guide nucleic acid sequence, and the variant exhibits one or more improved characteristics compared to the reference guide RNA.

[00640] Embodiment 42. The gNA variant of Embodiment 41, comprising a tracrRNA
stem loop comprising the sequence ¨UUU-N3-20-UUU¨ (SEQ ID NO: 4403).

[00641] Embodiment 43. The gNA variant of Embodiment 41 or 42, comprising a crRNA
sequence with ¨AAAG¨ in a location 5' to a targeting sequence of the gNA variant.

[00642] Embodiment 44. The gNA variant of Embodiment 43, wherein the ¨AAAG¨
sequence is immediately 5' to the targeting sequence.

[00643] Embodiment 45. The gNA variant of any one of Embodiments 41-44, wherein the gNA variant further comprises a targeting sequence wherein the targeting sequence is complementary to the target DNA sequence.

[00644] Embodiment 46. The gNA variant of any one of Embodiments 41- 45, wherein the one or more improved characteristics is selected from the group consisting of improved stability, improved solubility, improved resistance to nuclease activity, increased folding rate of the NA, decreased side product formation during folding, increased productive folding, improved binding affinity to a CasX protein, improved binding affinity to a target DNA, improved gene editing, and improved specificity.

[00645] Embodiment 47. The gNA variant of Embodiment 46, wherein the one or more of the improved characteristics of the CasX variant is at least about 1.1 to about 100,000-fold improved relative to the reference gNA of SEQ ID NO: 4 or SEQ ID NO: 5.

[00646] Embodiment 48. The CasX variant of Embodiment 46 or 47, wherein one or more of the improved characteristics of the CasX variant is at least about 10 to about 100-fold improved relative to the reference gNA of SEQ ID NO: 4 or SEQ ID NO: 5.

[00647] Embodiment 49. The gNA variant of any one of Embodiments 41-48, wherein the at least one modification comprises at least one nucleotide substitution in a region of the gNA
variant compared to the reference gNA of SEQ ID NO: 4 or SEQ ID NO: 5.

[00648] Embodiment 50. The gNA variant of Embodiment 41- 49, wherein the at least one modification comprises substitution of at least 1 to 4 nucleotides in a region of the gNA variant compared to the reference gNA of SEQ ID NO: 4 or SEQ ID NO: 5.

[00649] Embodiment 51. The gNA variant of any one of Embodiments 41- 50, wherein the at least one modification comprises at least one nucleotide deletion in a region of the gNA variant compared to the reference gNA of SEQ ID NO: 4 or SEQ ID NO: 5.

[00650] Embodiment 52. The gNA variant of Embodiments 41- 51, wherein the at least one modification comprises deletion of 1 to 4 nucleotides in a region of the gNA
variant compared to the reference gNA of SEQ ID NO: 4 or SEQ ID NO: 5.

[00651] Embodiment 53. The gNA variant of any one of Embodiments 41- 52, wherein the at least one modification comprises at least one nucleotide insertion in a region of the gNA variant compared to the reference gNA of SEQ ID NO: 4 or SEQ ID NO: 5.

[00652] Embodiment 54. The gNA variant of any one of Embodiments 41-53, wherein the at least one modification comprises insertion of 1 to 4 nucleotides in a region of the gNA variant compared to the reference gNA of SEQ ID NO: 4 or SEQ ID NO: 5.

[00653] Embodiment 55. The gNA variant of any one of Embodiments 41- 54, wherein the at least one modification comprises a deletion of at least 1 to 4 nucleotides, an insertion of at least 1 to 4 nucleotides, a substitution of at least 1 to 4 nucleotides, or any combination thereof in a region of the gNA variant compared to the reference gNA of SEQ ID NO: 4 or SEQ
ID NO: 5.

[00654] Embodiment 56. The gNA variant of any one of Embodiments 41- 5, comprising a scaffold region at least 60% homologous to SEQ ID NO: 4 or SEQ ID NO: 5.

[00655] Embodiment 57. The gNA variant of any one of Embodiments 41- 55, comprising a scaffold NA stem loop at least 60% homologous to SEQ ID NO: 14.

[00656] Embodiment 58. The gNA variant of any one of Embodiments 41- 55, comprising an extended stem loop at least 60% homologous to SEQ ID NO: 14.

[00657] Embodiment 59. The gNA variant of any one of Embodiments 41- 55, wherein the gNA variant sequence is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70%, or at least 80% homologous to SEQ ID NO: 4.

[00658] Embodiment 60. The gNA variant of any one of Embodiments 41-58, wherein the gNA variant sequence is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous, or is 100% homologous to a sequence selected from the group of sequences of SEQ ID NOS: 2101-2241.

[00659] Embodiment 61. The gNA variant of any one of Embodiments 41- 60, comprising an extended stem loop region comprising fewer than 10,000 nucleotides.

[00660] Embodiment 62. The gNA variant of any one of Embodiments 41-60, wherein the scaffold stem loop or the extended stem loop sequence is replaced with an exogenous stem loop sequence.

[00661] Embodiment 63. The gNA variant of Embodiment t 62, wherein the exogenous stem loop is a hairpin loop that is capable of binding a protein, RNA or DNA
molecule.

[00662] Embodiment 64. The gNA variant of Embodiment 62 or 63, wherein the exogenous stem loop is a hairpin loop that increases the stability of the gNA.

[00663] Embodiment 65. The gNA variant of Embodiment 63 or 64, wherein the hairpin loop is selected from M52, Qf3, U1A, or PP7.

[00664] Embodiment 66. The gNA variant of any one of Embodiments 41- 65, further comprising one or more ribozymes.

[00665] Embodiment 67. The gNA variant of Embodiment 66, wherein the one or more ribozymes are independently fused to a terminus of the gNA variant.

[00666] Embodiment 68. The gNA variant of Embodiment 66 or 67, wherein at least one of the one or more ribozymes are an hepatitis delta virus (HDV) ribozyme, hammerhead ribozyme, pistol ribozyme, hatchet ribozyme, or tobacco ringspot virus (TRSV) ribozyme.

[00667] Embodiment 69. The gNA variant of any one of Embodiments 41-68, further comprising a protein binding motif.

[00668] Embodiment 70. The gNA variant of any one of Embodiments 41-69, further comprising a thermostable stem loop.

[00669] Embodiment 71. The gNA variant of Embodiment 41, comprising the sequence of any one of SEQ ID NO: 2101-2241.

[00670] Embodiment 72. The gNA variant of any one of Embodiments 41-71, further comprising a targeting sequence.

[00671] Embodiment 73. The gNA variant of Embodiment 72, wherein the targeting sequence has 14, 15, 16, 18, 18, 19, 20, 21, 22, 23 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides.

[00672] Embodiment 74. The gNA variant of any one of Embodiments 41- 73, wherein the gNA is chemically modified.

[00673] Embodiment 75. A gene editing pair comprising a CasX protein and a first gNA.

[00674] Embodiment 76. The gene editing pair of Embodiment 74, wherein the first gNA
comprises:
a. a gNA variant of any one of Embodiments 41- 74 and a targeting sequence;
or b. a reference guide nucleic acid of SEQ ID NOS: 4 or 5 and a targeting sequence, wherein the targeting sequence is complementary to the target nucleic acid.

[00675] Embodiment 77. The gene editing pair of Embodiment 74 or 76, wherein the CasX
comprises:
a. a CasX variant of any one of Embodiments 1- 40; or b. a reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[00676] Embodiment 78. The gene editing pair of any one of Embodiments 74- 77, further comprising a second gNA or a nucleic acid encoding the second gNA, wherein the second gNA
has a targeting sequence complementary to a different portion of the target nucleic acid compared to the targeting sequence of the first gNA.

[00677] Embodiment 79. The gene editing pair of any one of Embodiments 74- 78, wherein the CasX protein and the gNA are capable of associating together in a ribonuclear protein complex (RNP).

[00678] Embodiment 80. The gene editing pair of any one of Embodiments 74-79, wherein the CasX protein and the gNA are associated together in a ribonuclear protein complex (RNP).

[00679] Embodiment 81. The gene editing pair of Embodiment 79 or 80, wherein the RNP is capable of binding a target DNA.

[00680] Embodiment 82. The gene editing pair of any one of Embodiments 79- 81, wherein the RNP has a higher percentage of cleavage-competent RNP compared to an RNP of a reference CasX protein and a reference guide nucleic acid.

[00681] Embodiment 83. The gene editing pair of any one of Embodiments 79- 82, wherein the RNP is capable of binding and cleaving a target DNA.

[00682] Embodiment 84. The gene editing pair of any one of Embodiments 79- 82, wherein the RNP binds a target DNA but does not cleave the target DNA.

[00683] Embodiment 85. The gene editing pair of any one of Embodiments 79- 83, wherein the RNP is capable of binding a target DNA and generating one or more single-stranded nicks in the target DNA.

[00684] Embodiment 86. The gene editing pair of any one of Embodiments 79-83 or 85, wherein the gene editing pair has one or more improved characteristics compared to a gene editing pair comprising a reference CasX protein of SEQ ID NO: 1, SEQ ID NO:
2, or SEQ ID
NO: 3 and a reference guide nucleic acid of SEQ ID NOS: 4 or 5.

[00685] Embodiment 87. The gene editing pair of Embodiment 86, wherein the one or more improved characteristics comprises improved CasX:gNA RNP complex stability, improved binding affinity between the CasX and gNA, improved kinetics of RNP complex formation, higher percentage of cleavage-competent RNP, improved RNP binding affinity to the target DNA, improved unwinding of the target DNA, increased editing activity, improved editing efficiency, improved editing specificity, increased activity of the nuclease, increased target strand loading for double strand cleavage, decreased target strand loading for single strand nicking, decreased off-target cleavage, improved binding of the non-target strand of DNA, or improved resistance to nuclease activity.

[00686] Embodiment 88. The gene editing pair of Embodiment 86 or 87, wherein the at least one or more of the improved characteristics is at least about 1.1 to about 100,000-fold improved relative to a gene editing pair of the reference CasX protein and the reference guide nucleic acid.

[00687] Embodiment 89. The gene editing pair of any one of Embodiments 86- 88, wherein one or more of the improved characteristics of the CasX variant is at least about 10 to about 100-fold improved relative to a gene editing pair of the reference CasX
protein and the reference guide nucleic acid.

[00688] Embodiment 90. A method of editing a target DNA, comprising contacting the target DNA with a gene editing pair of any one of Embodiments 74- 89, wherein the contacting results in editing of the target DNA.

[00689] Embodiment 91. The method of Embodiment 90, comprising contacting the target DNA with a plurality of gNAs comprising targeting sequences complementary to different regions of the target DNA.

[00690] Embodiment 92. The method of Embodiment 90 or 91, wherein the contacting introduces one or more single-stranded breaks in the target DNA and wherein the editing comprises a mutation, an insertion, or a deletion in the target DNA.

[00691] Embodiment 93. The method of Embodiment 90 or 91, wherein the contacting comprises introducing one or more double-stranded breaks in the target DNA and wherein the editing comprises a mutation, an insertion, or a deletion in the target DNA.

[00692] Embodiment 94. The method of any one of Embodiments 90- 93, further comprising contacting the target DNA with a nucleotide sequence of a donor template nucleic acid wherein the donor template comprises a nucleotide sequence having homology to the target DNA.

[00693] Embodiment 95. The method of Embodiment 94, wherein the donor template is inserted in the target DNA at the break site by homology-directed repair.

[00694] Embodiment 96. The method of any one of Embodiments 90- 95, wherein editing occurs in vitro outside of a cell.

[00695] Embodiment 97. The method of any one of Embodiments 90- 95, wherein editing occurs in vitro inside of a cell.

[00696] Embodiment 98. The method of any one of Embodiments 90- 95, wherein editing occurs in vivo inside of a cell.

[00697] Embodiment 99. The method of Embodiments 97 or 98, wherein the cell is a eukaryotic cell.

[00698] Embodiment 100. The method of Embodiment 99, wherein the eukaryotic cell is selected from the group consisting of a plant cell, a fungal cell, a protist cell, a mammalian cell, a reptile cell, an insect cell, an avian cell, a fish cell, a parasite cell, an arthropod cell, a cell of an invertebrate, a cell of a vertebrate, a rodent cell, a mouse cell, a rat cell, a primate cell, a non-human primate cell, and a human cell.

[00699] Embodiment 101. The method of Embodiment 99 or 100, wherein the method comprises contacting the eukaryotic cell with a vector encoding or comprising the CasX protein and the gNA, and optionally further comprising the donor template.

[00700] Embodiment 102. The method of Embodiment 101, wherein the vector is an Adeno-Associated Viral (AAV) vector.

[00701] Embodiment 103. The method of Embodiment 102, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-Rh74, or AAVRh10.

[00702] Embodiment 104. The method of Embodiment 101, wherein the vector is a lentiviral vector.

[00703] Embodiment 105. The method of Embodiment 101, wherein the vector is a virus-like particle (VLP).

[00704] Embodiment 106. The method of any one of Embodiments 101- 105, wherein the vector is administered to a subject at a therapeutically effective dose.

[00705] Embodiment 107. The method of Embodiment 105, wherein the subject is selected from the group consisting of mouse, rat, pig, non-human primate, and human.

[00706] Embodiment 108. The method of Embodiment 107, wherein the subject is a human.

[00707] Embodiment 109. The method of any one of Embodiments 106- 108, wherein the vector is administered at a dose of at least about 1 x 1010 vector genomes (vg), or at least about 1 x 1011 vg, or at least about 1 x 1012 vg, or at least about 1 x 1013 vg, or at least about 1 x 1014 vg, or at least about 1 x 1015 vg, or at least about 1 x 1016 vg.

[00708] Embodiment 110. The method of any one of Embodiments 106- 109, wherein the vector is administered by a route of administration selected from the group consisting of intraparenchymal, intravenous, intra-arterial, intracerebroventricular, intraci sternal, intrathecal, intracranial, and intraperitoneal routes.

[00709] Embodiment 111. The method of Embodiment 97, wherein the cell is a prokaryotic cell.

[00710] Embodiment 112. A cell comprising a CasX variant, wherein the CasX
variant is a CasX variant of any one of Embodiments 1-40.

[00711] Embodiment 113. The cell of Embodiment 112, further comprising a. a gNA variant of any one of Embodiments 41- 74, or b. a reference guide nucleic acid of SEQ ID NOS: 4 or 5 and a targeting sequence.

[00712] Embodiment 114. A cell comprising a gNA variant of any one of Embodiments 41-74.

[00713] Embodiment 115. The cell of Embodiment 114, further comprising a CasX
variant of any one of Embodiments 1 to Embodiment 35, or a CasX protein of SEQ ID NO: 1, SEQ ID
NO: 2, or SEQ ID NO. 3.

[00714] Embodiment 116. The cell of Embodiment 114 or 115, further comprising a donor nucleotide template comprising a sequence that hybridizes with a target DNA.

[00715] Embodiment 117. The cell of Embodiment 116, wherein the donor template ranges in size from 10-10,000 nucleotides.

[00716] Embodiment 118. The cell of Embodiment 116 or 117, wherein the donor template is a single-stranded DNA template or a single stranded RNA template.

[00717] Embodiment 119. The method of Embodiment 116 or 117, wherein the donor template is a double-stranded DNA template.

[00718] Embodiment 120. The cell of any one of Embodiments 112- 119, wherein the cell is a eukaryotic cell.

[00719] Embodiment 121. The cell of any one of Embodiments 112- 119, wherein the cell is a prokaryotic cell.

[00720] Embodiment 122. A polynucleotide encoding the CasX variant of any one of Embodiments 1 to 40.

[00721] Embodiment 123. A polynucleotide encoding the gNA variant of any one of Embodiments 41- 74.

[00722] Embodiment 124. A vector comprising the polynucleotide of Embodiment 122 and/or 123.

[00723] Embodiment 125. The vector of Embodiment 123, wherein the vector is an Adeno-Associated Viral (AAV) vector.

[00724] Embodiment 126. The method of Embodiment 125, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-Rh74, or AAVRh10.

[00725] Embodiment 127. The vector of Embodiment 123, wherein the vector is a lentiviral vector.

[00726] Embodiment 128. The vector of Embodiment 124, wherein the vector is a virus-like particle (VLP).

[00727] Embodiment 129. A cell comprising the polynucleotide of Embodiment 122, or the vector of any one of Embodiments 124-128.

[00728] Embodiment 130. A composition, comprising the CasX variant of any one of Embodiments 1 to 35.

[00729] Embodiment 131. The composition of Embodiment 130, further comprising:
a. a gNA variant of any one of Embodiments 45- 74, or b. the reference guide RNA of SEQ ID NOS: 4 or 5 and a targeting sequence.

[00730] Embodiment 132. The composition of Embodiment 130 or 131, wherein the CasX
protein and the gNA are associated together in a ribonuclear protein complex (RNP).

[00731] Embodiment 133. The composition of any one of Embodiments 130- 132, further comprising a donor template nucleic acid wherein the donor template comprises a nucleotide sequence having homology to a target DNA.

[00732] Embodiment 134. The composition of any one of Embodiments 130-133, further comprising a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

[00733] Embodiment 135. A composition, comprising a gNA variant of any one of Embodiments 41- 74.

[00734] Embodiment 136. The composition of Embodiment 135, further comprising the CasX
variant of any one of Embodiments 1 to 35, or the CasX protein of SEQ ID NO:
1, SEQ ID NO:
2, or SEQ ID NO: 3.

[00735] Embodiment 137. The composition of Embodiment 136, wherein the CasX
protein and the gNA are associated together in a ribonuclear protein complex (RNP).

[00736] Embodiment 138. The composition of any one of Embodiments 135- 137, further comprising a donor template nucleic acid wherein the donor template comprises a nucleotide sequence having homology to a target DNA.

[00737] Embodiment 139. The composition of any one of Embodiments 135- 138, further comprising a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

[00738] Embodiment 140. A composition, comprising the gene editing pair of any one of Embodiments 4- 89.

[00739] Embodiment 141. The composition of Embodiment 140, further comprising a donor template nucleic acid wherein the donor template comprises a nucleotide sequence having homology to a target DNA.

[00740] Embodiment 142. The composition of Embodiment 140 or 141, further comprising a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

[00741] Embodiment 143. A kit, comprising the CasX variant of any one of Embodiments 1 to 35 and a container.

[00742] Embodiment 144. The kit of Embodiment 143, further comprising:
a. a gNA variant of any one of Embodiments 45- 74, or b. the reference guide RNA of SEQ ID NOS: 4 or 5 and a targeting sequence.

[00743] Embodiment 145. The kit of Embodiment 143 or 144, further comprising a donor template nucleic acid wherein the donor template comprises a nucleotide sequence having homology to a target sequence of a target DNA.

[00744] Embodiment 146. The kit of any one of Embodiments 143-145, further comprising a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

[00745] Embodiment 147. A kit, comprising a gNA variant of any one of Embodiments 45- 74.

[00746] Embodiment 148. The kit of Embodiment 147, further comprising the CasX
variant of any one of Embodiments 1 to 35, or the CasX protein of SEQ ID NO: 1, SEQ ID
NO: 2, or SEQ
ID NO: 3.

[00747] Embodiment 149. The kit of Embodiment 147 or 148, further comprising a donor template nucleic acid wherein the donor template comprises a nucleotide sequence having homology to a target sequence of a target DNA.

[00748] Embodiment 150. The kit of any one of Embodiments 147-149, further comprising a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

[00749] Embodiment 151. A kit, comprising the gene editing pair of any one of Embodiments 74-89.

[00750] Embodiment 152. The kit of Embodiment 151, further comprising a donor template nucleic acid wherein the donor template comprises a nucleotide sequence having homology to a target DNA.

[00751] Embodiment 153. The kit of Embodiment 151 or 152, further comprising a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

[00752] Embodiment 154. A CasX variant comprising any one of the sequences listed in Table 3.

[00753] Embodiment 155. A gNA variant comprising any one of the sequences listed in Table 2.

[00754] Embodiment 156. The gNA variant of Embodiment 155, further comprising a targeting sequence of at least 10 to 30 nucleotides complementary to a target DNA.

[00755] Embodiment 157. The gNA variant of Embodiment 156, wherein the targeting sequence has 20 nucleotides.

[00756] Embodiment 158. The gNA variant of Embodiment 156, wherein the targeting sequence has 19 nucleotides.

[00757] Embodiment 159. The gNA variant of Embodiment 156, wherein the targeting sequence has 18 nucleotides

[00758] Embodiment 160. The gNA variant of Embodiment 156, wherein the targeting sequence has 17 nucleotides

[00759] Embodiment 161. The CasX variant of any one of Embodiments 1 to 40, wherein the CasX protein comprises a first domain from a first CasX protein and second domain from a second CasX protein different from the first CasX protein.

[00760] Embodiment 162. The CasX variant of Embodiment 161, wherein the first domain is selected from the group consisting of the NTSB, TSL, helical I, helical II, OBD, and RuvC
domains.

[00761] Embodiment 163. The CasX variant of Embodiment 162, wherein the second domain is selected from the group consisting of the NTSB, TSL, helical I, helical II, OBD, and RuvC
domains.

[00762] Embodiment 164. The CasX variant of any one of Embodiments 161 163, wherein the first and second domains are not the same domain.

[00763] Embodiment 165. The CasX variant of any one of Embodiments 161- 164 wherein the first CasX protein comprises a sequence of SEQ ID NO: 1 and the second CasX
protein comprises a sequence of SEQ ID NO: 2.

[00764] Embodiment 166. The CasX variant of any one of Embodiments 161-164 wherein the first CasX protein comprises a sequence of SEQ ID NO: 1 and the second CasX
protein comprises a sequence of SEQ ID NO: 3.

[00765] Embodiment 167. The CasX variant of any one of Embodiments 161-164, wherein the first CasX protein comprises a sequence of SEQ ID NO: 2 and the second CasX
protein comprises a sequence of SEQ ID NO: 3.

[00766] Embodiment 168. The CasX variant of any one of Embodiments 1 to 40 or 161- 167, wherein the CasX protein comprises at least one chimeric domain comprising a first part from a first CasX protein and a second part from a second CasX protein different from the first CasX
protein.

[00767] Embodiment 169. The CasX variant of Embodiment 168, wherein the at least one chimeric domain is selected from the group consisting of the NTSB, TSL, helical I, helical II, OBD, and RuvC domains.

[00768] Embodiment 170. The CasX variant of Embodiment 168 or 169, wherein the first CasX protein comprises a sequence of SEQ ID NO: 1 and the second CasX protein comprises a sequence of SEQ ID NO: 2.

[00769] Embodiment 171. The CasX variant of Embodiment 168 or 169, wherein the first CasX protein comprises a sequence of SEQ ID NO: 1 and the second CasX protein comprises a sequence of SEQ ID NO: 3.

[00770] Embodiment 172. The CasX variant of Embodiment 168 or 169, wherein the first CasX protein comprises a sequence of SEQ ID NO: 2 and the second CasX protein comprises a sequence of SEQ ID NO: 3.

[00771] Embodiment 173. The CasX variant of Embodiment 168, wherein the at least one chimeric domain comprises a chimeric RuvC domain.

[00772] Embodiment 174. The CasX variant of Embodiment 173, wherein the chimeric RuvC
domain comprises amino acids 661 to 824 of SEQ ID NO: 1 and amino acids 922 to 978 of SEQ
ID NO: 2.

[00773] Embodiment 175. The CasX variant of Embodiment 173, wherein the chimeric RuvC
domain comprises amino acids 648 to 812 of SEQ ID NO: 2 and amino acids 935 to 986 of SEQ
ID NO: 1.

[00774] Embodiment 176. The gNA variant of any one of Embodiments 41-74, wherein the gNA comprises a first region from a first gNA and a second region from a second gNA.

[00775] Embodiment 177. The gNA variant of Embodiment 176, wherein the first region is selected from the group consisting of a triplex region, a scaffold stem loop, and an extended stem loop.

[00776] Embodiment 178. The gNA variant of Embodiment 176 or 177, wherein the second region is selected from the group consisting of a triplex region, a scaffold stem loop, and an extended stem loop.

[00777] Embodiment 179. The gNA variant of any one of Embodiments 176- 178, wherein the first and second regions are not the same region.

[00778] Embodiment 180. The gNA variant of any one of Embodiments 176- 179, wherein the first gNA comprises a sequence of SEQ ID NO: 4 and the second gNA comprises a sequence of SEQ ID NO: 5.

[00779] Embodiment 181. The gNA variant of any one of Embodiments 41- 74 or 176- 180, comprising at least one chimeric region comprising a first part from a first gNA and a second part from a second gNA.

[00780] Embodiment 182. The gNA variant of Embodiment 181, wherein the at least one chimeric region is selected from the group consisting of a triplex region, a scaffold stem loop, and an extended stem loop.

[00781] Embodiment 183. The gNA variant of Embodiment 182, wherein the first gNA
comprises a sequence of SEQ ID NO: 4 and the second gNA comprises a sequence of SEQ ID
NO: 5.

[00782] The following Examples are merely illustrative and are not meant to limit any aspects of the present disclosure in any way.
EXAMPLES
Example 1: Assays used to measure sgRNA and CasX protein activity

[00783] Several assays were used to carry out initial screens of CasX protein and sgRNA DME
libraries and engineered mutants, and to measure the activity of select protein and sgRNA
variants relative to CasX reference sgRNAs and proteins.
E. coil CRISPRi screen:

[00784] Briefly, biological triplicates of dead CasX DME Libraries on a chloramphenicol (CM) resistant plasmid with a GFP guide RNA on a carbenicillin (Carb) resistant plasmid were transformed (at > 5x library size) into MG1655 with genetically integrated and constitutively expressed GFP and RFP (see FIG. 13A-13B). Cells were grown overnight in EZ-RDM
+ Carb, CM and Anhydrotetracycline (aTc) inducer. E. coil were FACS sorted based on gates for the top 1% of GFP but not RFP repression, collected, and resorted immediately to further enrich for highly functional CasX molecules. Double sorted libraries were then grown out and DNA was collected for deep sequencing on a highseq. This DNA was also re-transformed onto plates and individual clones were picked for further analysis.
E.coli Toxin selection:

[00785] Briefly carbenicillin resistant plasmid containing an arabinose inducible toxin were transformed into E.coli cells and made electrocompetent. Biological triplicates of CasX DME
Libraries with a toxin targeted guide RNA on a chloramphenicol resistant plasmid were transformed (at > 5x library size) into said cells and grown in LB + CM and arabinose inducer.
E. coil that cleaved the toxin plasmid survived in the induction media and were grown to mid log and plasmids with functional CasX cleavers were recovered. This selection was repeated as needed. Selected libraries were then grown out and DNA was collected for deep sequencing on a highseq. This DNA was also re-transformed onto plates and individual clones were picked for further analysis and testing.
Lentiviral based screen EGFP screen:

[00786] Lentiviral particles were produced in HEK293 cells at a confluency of 70%-90% at time of transfection. Cells were transfected using polyethylenimine based transfection of plasmids containing a CasX DME library. Lentiviral vectors were co-transfected with the lentiviral packaging plasmid and the VSV-G envelope plasmids for particle production. Media was changed 12 hours post-transfection, and virus harvested at 36-48 hours post-transfection.
Viral supernatants were filtered using 0.45mm membrane filters, diluted in cell culture media if appropriate, and added to target cells HEK cells with an Integrated GFP
reporter. Polybrene was supplemented to enhance transduction efficiency, if necessary. Transduced cells were selected for 24-48 hours post-transduction using puromycin and grown for 7-10 days.
Cells were then sorted for GFP disruption & collected for highly functional sgRNA or protein variants (see FIG.
2). Libraries were then Amplified via PCR directly from the genome and collected for deep sequencing on a highseq. This DNA could also be re-cloned and re-transformed onto plates and individual clones were picked for further analysis.
Assaying editing efficiency of an HEK EGFP reporter:

[00787] To assay the editing efficiency of CasX reference sgRNAs and proteins and variants thereof, EGFP HEK293T reporter cells were seeded into 96-well plates and transfected according to the manufacturer's protocol with lipofectamine 3000 (Life Technologies) and 100-200ng plasmid DNA encoding a reference or variant CasX protein, P2A¨puromycin fusion and the reference or variant sgRNA. The next day cells were selected with 1.5m/m1 puromycin for 2 days and analyzed by fluorescence-activated cell sorting (FACS) 7 days after selection to allow for clearance of EGFP protein from the cells. EGFP disruption via editing was traced using an Attune NxT Flow Cytometer and high-throughput autosampler.
Example 2: Cleavage efficiency of CasX reference sgRNA

[00788] The reference CasX sgRNA of SEQ ID NO: 4 (below) is described in WO
2018/064371, the contents of which are incorporated herein by reference.

UCGUAUGGAC
61 GAAGCGCUUA UUUAUCGGAG AGAAACCGAU AAGUAAAACG CAUCAAAG (SEQ ID NO: 4).

[00789] It was found that alterations to the sgRNA reference sequence of SEQ
ID NO: 4, producing SEQ ID NO: 5 (below) were able to improve CasX cleavage efficiency.

CGUAUGGGUA
61 AAGCGCUUAU UUAUCGGAGA GAAAUCCGAU AAAUAAGAAG CAUCAAAG (SEQ ID NO: 5).

[00790] To assay the editing efficiency of CasX reference sgRNAs and variants thereof, EGFP
HEK293T reporter cells were seeded into 96-well plates and transfected according to the manufacturer's protocol with lipofectamine 3000 (Life Technologies) and 100-200ng plasmid DNA encoding a reference CasX protein, P2A¨puromycin fusion and the sgRNA. The next day cells were selected with 1.5m/m1 puromycin for 2 days and analyzed by fluorescence-activated cell sorting (FACS) 7 days after selection to allow for clearance of EGFP
protein from the cells.
EGFP disruption via editing was traced using an Attune NxT Flow Cytometer and high-throughput autosampler.

[00791] When testing cleavage of an EGFP reporter by CasX reference and sgRNA
variants, the following DNA encoding spacer target sequences were used:
E6 (TGTGGTCGGGGTAGCGGCTG; SEQ ID NO: 29) and E7 (TCAAGTCCGCCATGCCCGAA; SEQ ID NO: 30).

[00792] An example of the increased cleavage efficiency of the sgRNA of SEQ ID
NO: 5 compared to the sgRNA of SEQ ID NO: 4 is shown in FIG. 5A. Editing efficiency of SEQ ID

NO: 5 was improved 176% compared to SEQ ID NO: 4. Accordingly, SEQ ID NO: 5 was chosen as reference sgRNA for DME and additional sgRNA variant design, described below.
Example 3: Mutagenesis of CasX reference gRNA produces variants with improved target cleavage

[00793] DME of the sgRNA was achieved using two distinct PCR methods. The first method, which generates single nucleotide substitutions, makes use of degenerate oligonucleotides.
These are synthesized with a custom nucleotide mix, such that each locus of the primer that is complementary to the sgRNA locus has a 97% chance of being the wild type base, and a 1%
chance of being each of the other three nucleotides. During PCR, the degenerate oligos anneal to, and just beyond, the sgRNA scaffold within a small plasmid, amplifying the entire plasmid.
The PCR product was purified, ligated, and transformed into E. coil. The second method was used to generate sgRNA scaffolds with single or double nucleotide insertions and deletions. A
unique PCR reaction was set up for each base pair intended for mutation: In the case of the CasX
scaffold of SEQ ID NO: 5, 109 PCRs were used. These PCR primers were designed and paired such that PCR products either were missing a base pair, or contained an additional inserted base pair. For inserted base pairs, PCR primers inserted a degenerate base such that all four possible nucleotides were represented in the final library.

[00794] Once constructed, both the protein and sgRNA DME libraries were assayed in a screen or selection as described in Example 1 to quantitatively identify mutations conferring enhanced functionality. Any assay, such as cell survival or fluorescence intensity, is sufficient so long as the assay maintains a link between genotype and phenotype. High throughput sequencing of these populations and validating individual variant phenotypes provided information about mutations that affect functionality as assayed by screening or selection.
Statistical analysis of deep sequencing data provided detailed insight into the mutation landscape and mechanism of protein function or guide RNA function (see FIG. 3A-3B, FIG. 4A, FIG. 4B, FIG.
4C).

[00795] DME libraries sgRNA RNA variants were made using a reference gRNA of SEQ ID
NO: 5, underwent selection or enrichment, and were sequenced to determine the fold enrichment of the sgRNA variants in the library. The libraries included every possible single mutation of every nucleotide, and double indels (insertion/deletions). The results are shown in FIGs. 3A-3B, FIG. 4A-4C, and Table 4 below.

[00796] To create a library of base pair substitutions using DME, two degenerate oligonucleotides that each bind to half of the sgRNA scaffold and together amplify the entire plasmid comprising the starting sgRNA scaffold were designed. These oligos were made from a custom nucleotide mix with a 3% mutation rate. These degenerate oligos were then used to PCR
amplify the starting scaffold plasmid using standard manufacturing protocols.
This PCR product was gel purified, again following standard protocols. The gel purified PCR
product was then blunt end ligated and electroporated into an appropriate E. coil cloning strain. Transformants were grown overnight on standard media, and plasmid DNA was purified via miniprep.

[00797] To generate a library of small insertions and deletions, PCR primers were designed such that the PCR products resulting from amplification of the plasmid comprising the base sgRNA scaffold would either be missing a base pair, or contain an additional inserted base pair.
For inserted base pairs, PCR primers were designed in which a degenerate base has been inserted, such that all four possible nucleotides were represented in the final library of pooled PCR products. The starting sgRNA scaffold was then PCR amplified with each set of oligos as their own reaction. Each PCR reaction contained five possible primers, although all primers annealed to the same sequence. For example, Primer 1 omitted a base, in order to create a deletion. Primers 2, 3, 4, and 5 inserted either an A, T, G, or C. However, these five primers all annealed to the same region and hence could be pooled in a single PCR.
However, PCRs for different positions along the sgRNA needed to be kept in separate tubes, and 109 distinct PCR
reactions were used to generate the sgRNA DME library.

[00798] The resulting 109 PCR products were then run on an agarose gel and excised before being combined and purified. The pooled PCR products were blunt ligated and electroporated into E. coil. Transformants were grown overnight on standard media with an appropriate selectable marker, and plasmid DNA was purified via miniprep. Having created a library of all single small indels, the steps of PCR amplifying the starting plasmid with each set of oligos, purifying, blunt end ligating, transforming into E. coil and mini-prepping can be repeated to obtain a library containing most double small indels. Combining the single indel library and double indel library at a ratio of 1:1000 resulted in a library that represented both single and double indels.

[00799] The resulting libraries were then combined and passed through the DME
screening and/or selection process to identify variants with enhanced cleavage activity.
DME libraries were screened using toxin cleavage and CRISPRi repression in E. coil, as well as EGFP cutting in lentiviral-transfected HEK293 cells, as described in Example 1. The fold enrichment of scaffold variants in DME libraries that have undergoing screening/selection followed by sequencing is shown below in Table 4. The read counts associated with each of the below sequences in Table 4 were determined ('annotations', 'seq'). Only sequences with at least 10 reads across any sample were analyzed to filter from 15 Million to 600 K sequences.
The below 'seq' gives the sequence of the entire insert between the two 5' random 5mer and the 3' random 5mer.
'seq short' gives the anticipated sequence of the scaffold only. The mutations associated with each sequence were determined through alignment ('muts'). All modifications are indicated by their [position (0-indexed)].[reference base].[alternate base]. Position 0 indicates the first T of the transcribed gRNA. Sequences with multiple mutations are semicolon separated. The column muts lindexed, gives the same information but 1-indexed instead of 0-indexed.
Each of the modifications are annotated ('annotated variants'), as being a single substitution/insertion/
deletion, double substitution/insertion/deletion, single del single sub (a deletion and an adjacent substitution), a single sub single ins (a substitution and adjacent insertion), 'outside ref (indicates that the modification is outside the transcribed gRNA), or 'other' (any larger substitution/insertion/deletion or some combination thereof). An insertion at position i indicates an inserted base between position i-1 and i (i.e. before the indicated position). To note about variant annotation: a deletion of any one of a consecutive set of bases can be attributed to any of those bases. Thus, a deletion of the T at position -1 is the same sequence as a deletion of the T at position 0. 'counts' indicates the sequencing-depth normalized read count per sequence per sample. Technical replicates were combined by taking the geometric mean.
'log2enrichment' gives the median enrichment (using a pseudocount of 10) across each context, or across all samples, after merging for technical replicates. The naive read count was averaged (geometric) between the D2 _N and D3 _N samples. Finally, the 'log2enrichment err' gives the 'confidence interval' on the mean 1og2 enrichment. It is the standard deviation of the enrichment across samples *2 / sqrt of the number of samples. Below, only the sequences with median log2enrichment - log2enrichment err > 0 are shown (2704/614564 sequences examined).

[00800] In Table 4, CI indicates confidence interval and MI indicates median enrichment, which indicates enhanced activity.
Table 4. Median Enrichment of DME Scaffold Variants index SEQ ID muts lindexed index SEQ
ID muts lindexed MI 95% CI

NO NO

7240543 412 27.-.C;76.G.- 3.390 2.040 2720034 439 2.A.C;0.T.-;78.-.0 2.531 0.492 n.) o 7240150 413 27.-.C;75.-.0 3.111 1.862 2265581 440 0.T.-;86.-.0 2.520 0.504 n.) o iz..1 2584994 414 0.T.-;2.A.C;27.-.0 2.997 1.806 2256355 441 0.T.-;76.GG.-C 2.516 0.942 .6.
--.1 oe 2618163 415 0.T.-;2.A.C;55.-.G 2.915 0.725 7251229 442 27.-.C;76.-.G 2.516 1.793 oe n.) 2655870 416 2.A.C;0.T.-;76.GG.-A 2.903 0.391 10281529 443 17.-.T;76.GG.-A 2.515 1.104 2762330 417 2.A.C;0.T.-;55.-.T 2.857 1.290 2299702 444 0.T.-;74.-.T 2.504 0.392 7247368 418 27.-.C;86.C.- 2.835 1.637 2670445 445 2.A.C;0.T.-;85.T.- 2.499 1.225 2731505 419 2.A.C;0.T.-;75.-.G 2.795 0.625 2258816 446 0.T.-;76.G.- 2.494 0.475 2729600 420 2.A.C;0.T.-;76.-.T 2.791 0.628 7241311 447 27.-.C;77.GA.-- 2.493 1.595 2701142 421 2.A.C;0.T.-;87.-.T 2.768 0.559 2658150 448 2.A.C;0.T.-;76.GG.-C 2.492 0.585 2659588 422 2.A.C;0.T.-;75.-.0 2.733 0.477 2734378 449 2.A.C;0.T.-;74.-.T 2.490 0.485 P
2582823 423 0.T.-;2.A.C;27.-.A 2.729 1.669 2723181 450 2.A.C;0.T.-;76.-.G 2.488 0.421 ,..
, r., n.) 3000598 424 1.TA.--;76.G.-2.704 0.439 2288202 451 0.T.-;81.GA.-T 2.487 0.591 1--, .
,..
--.1 10565036 425 15.-.T;74.-.T 2.681 0.808 2278172 452 0.T.-;89.-.0 2.486 0.690 "
r., ,--µ, 9696472 426 28.-.T;76.GG.-T 2.681 1.715 2997382 453 1.TA.--;76.GG.-A 2.465 1.066 , r., , 2674674 427 2.A.C;0.T.-;86.-.0 2.650 0.772 2255017 454 0.T.-;76.GG.-A 2.463 0.422 .
7254130 428 27.-.C;75.CG.-T 2.629 1.755 2257399 455 0.T.-;75.-.0 2.460 0.676 2977442 429 1.TA.--;55.-.G 2.629 0.887 12183183 456 2.A.-;81.GA.-T 2.459 0.736 2661951 430 2.A.C;0.T.-;76.G.- 2.627 0.432 7252067 457 27.-.C;76.GG.-T 2.459 2.062 1937646 431 2.A.C;0.TT.--;75.-.0 2.626 1.328 10525083 458 15.-.T;75.-.0 2.448 1.006 2232796 432 0.T.-;55.-.G 2.607 0.777 7253869 459 27.-.C;74.-.T 2.439 1.638 2714418 433 0.T.-;2.A.C;81.GA.-T 2.595 0.443 4303777 460 4.T.-;76.-.T 2.435 0.782 IV
n 2700142 434 2.A.C;0.T.-;87.-.G 2.582 0.608 2741395 461 2.A.C;0.T.-;73.A.- 2.435 0.633 1-3 2667512 435 2.A.C;0.T.-;77.GA.-- 2.577 0.588 7250940 462 27.-.C;78.A.- 2.423 2.064 cp n.) o 7239606 436 27.-.C;76.-.A 2.566 1.441 4302595 463 4.T.-;76.GG.-T 2.422 0.850 n.) o 10563356 437 15.-.T;75.-.G 2.557 1.056 4275786 464 4.T.-;87.-.T 2.420 1.019 c,.) o 7181049 438 27.-.A;75.-.0 2.543 1.893 2650980 465 2.A.C;0.T.-;74.-.0 2.414 0.462 un =
un index SEQ ID muts lindexed index SEQ
ID muts lindexed MI 95% CI
MI 95% CI
NO NO

2458336 466 1.TA.--;3.C.A;76.G.- 2.411 1.089 2253698 492 0.T.-;75.-.A 2.334 0.918 n.) o 10284144 467 17.-.T;76.G.- 2.406 1.638 2468003 493 1.TA.--;3.C.A;75.-.G 2.330 0.934 n.) o iz..1 2726809 468 2.A.C;0.T.-;76.G.-;78.A.T 2.400 0.556 12290253 494 2.A.-;28.-.0 2.326 1.588 .6.
--.1 oe 2280896 469 0.T.-;87.-.T 2.398 0.560 2999382 495 1.TA.--;75.-.0 2.315 0.592 oe n.) 2673790 470 2.A.C;0.T.-;88.G.- 2.398 1.017 3227871 496 2.A.G;0.T.-;55.-.G 2.314 0.774 3188700 471 0.T.-;2.A.G;27.-.0 2.394 1.732 10521017 497 15.-.T;74.-.0 2.314 0.910 9632434 472 16. 10089663 498 19.-.T;75.-.G 2.308 1.078 2.394 1.141 .CTCATTACTTTG;75.-.G
4274894 499 4.T.-;87.-.G 2.308 0.512 3029757 473 1.TA.--;78.A.- 2.392 0.524 2466567 500 1.TA.--;3.C.A;78.A.- 2.308 1.291 2728393 474 2.A.C;0.T.-;76.GG.-T 2.390 0.714 2696261 501 2.A.C;0.T.-;89.-.0 2.293 0.681 2300381 475 0.T.-;75.CG.-T 2.385 0.948 2675948 502 2.A.C;0.T.-;89.-.A 2.289 1.259 P
2279969 476 0.T.-;86.C.-2.382 0.404 .
10521784 503 15.-.T;74.-.G 2.283 0.905 ,..
, 2260011 477 0.T.;77..0 2.379 0.608 .
"
n.) - -12123787 504 2.A.-;76.G.- 2.278 0.492 ' 1--, .3 oe 2248579 478 0.T.-;72.-.0 2.377 0.743 ,..
10310335 505 17.-.T;76.GG.-T 2.275 0.804 "
.
12075394 479 2.A.-;55.-.G
2.377 0.679 IV
I--`
2295876 506 0.T.-;77.-.T 2.273 0.931 , , 9602743 480 28.-.C;76.GG.-C
2.376 1.681 N, , 2697871 507 0.T.-;2.A.C;89.-.T 2.250 0.626 .
2736722 481 2.A.C;0.T.-;73.AT.-C 2.374 1.104 2735417 508 2.A.C;0.T.-;75.CG.-T 2.249 0.390 12117240 482 2.A.-;76.GG.-A 2.372 0.429 2671836 509 0.T.-;2.A.C;86.-.A 2.245 0.542 10307397 483 17.-.T;78.-.0 2.365 0.868 12033345 510 2.A.-;27.-.0 2.235 1.903 3034775 484 1.TA.--;75.-.G 2.360 0.992 2821484 511 0.T.-;2.A.C;17.-.T 2.235 0.750 12030812 485 2.A.-;27.-.A 2.355 1.651 3033813 512 1.TA.--;76.-.T 2.229 0.548 10530683 486 15.-.T;86.-.A 2.355 0.999 2291551 513 0.T.-;78.-.0 2.226 0.532 IV
12202799 487 2.A.-;75.-.G
2.352 0.508 n 2716457 514 2.A.C;0.T.-;80.A.- 2.213 0.548 1-3 9687168 488 28.-.T;76.GG.-A 2.351 1.612 2697599 515 2.A.C;0.T.-;89.A.- 2.209 1.346 cp n.) 4309853 489 4.T.-;75.CG.-T 2.344 0.845 12135440 516 2.A.-;87.-.A 2.208 1.053 2 o 4234320 490 4.T.-;75.-.0 2.344 0.820 -1 4273350 517 4.T.-;88.-.T 2.208 1.013 c,.) 2698521 491 2.A.C;0.T.-;88.-.T
2.339 0.685 o un 2298121 518 0.T.-;75.-.G 2.208 0.241 =
un index SEQ ID muts lindexed index SEQ
ID muts lindexed MI 95% CI
MI 95% CI
NO NO

2652510 519 0.T.-;2.A.C;74.-.G 2.206 0.613 12168820 546 2.A.-;87.-.T 2.140 0.458 n.) o 3006640 520 1.TA.--;86.-.0 2.206 0.584 2466824 547 1.TA.--;3.C.A;76.-.G 2.137 0.989 n.) o iz..1 10313388 521 17.-.T;74.-.T 2.206 1.036 3036963 548 1.TA.--;75.CG.-T 2.137 0.479 .6.
--.1 oe 10081410 522 19.-.T;87.-.G 2.206 0.589 10522450 549 15.-.T;75.-.A 2.135 1.003 oe n.) 3033236 523 1.TA.--;76.GG.-T 2.198 0.669 10300736 550 17.-.T;87.-.T 2.134 1.348 7242523 524 27.-.C;86.-.0 2.198 1.973 3002220 551 1.TA.--;79.G.- 2.131 0.607 7254383 525 27.-.C;73.AT.-C 2.198 1.510 3030471 552 .. 1.TA.--;76.-.G .. 2.130 .. 0.372 2264531 526 0.T.-;87.-.A 2.198 0.778 10523429 553 15.-.T;76.GG.-A 2.130 0.787 2727301 527 0.T.-;2.A.C;77.-.T 2.197 1.323 1909254 554 0.TTA.---;3.C.A;75.-.G 2.130 1.147 3019306 528 1.TA.--;87.-.G 2.191 0.534 3004722 555 1.TA.--;85.T.- 2.124 1.092 4295725 529 4.T.-;78.A.- 2.187 0.609 2672731 556 2.A.C;0.T.-;87.-.A 2.121 0.898 P
10311816 530 17.-.T;75.-.G 2.187 1.507 12129733 557 2.A.-;77.GA.-- 2.120 0.500 ,..
, r., n.) 12167745 531 2.A.-;87.-.G
2.184 0.736 4250089 558 4.T.-;89.-.A 2.117 0.998 1--, .
,..
o 12199256 532 2.A.-;76.GG.-T 2.179 0.737 2688981 559 2.A.C;0.T.-;99.-.G 2.112 0.980 "
r., ,--µ, 6477911 533 16.-.C;75.-.G 2.178 0.983 2995452 560 1.TA.--;74.-.G 2.112 0.611 , r., , 4274124 534 4.T.-;86.C.- 2.171 0.474 12114782 561 2.A.-;75.-.A 2.110 0.500 .
12206105 535 2.A.-;74.-.T 2.170 0.608 2993173 562 1.TA.--;73.-.A 2.104 0.697 12166825 536 2.A.-;86.C.- 2.168 0.774 1978344 563 0.T.C;87.-.G 2.100 0.870 11956698 537 2.AC.--;4.T.C;86.-.0 2.164 1.360 4294004 564 4.T.-;78.-.0 2.099 0.595 2280390 538 0.T.-;87.-.G 2.162 0.479 10568306 565 15.-.T;73.A.- 2.096 0.741 2650159 539 2.A.C;0.T.-;74.T.- 2.161 0.517 10561545 566 15.-.T;76.GG.-T 2.095 0.554 10531253 540 15.-.T;87.-.A 2.159 1.130 2713433 567 2.A.C;0.T.-;82.AA.-T 2.094 0.560 IV
n 2665054 541 2.A.C;0.T.-;79.G.- 2.158 0.562 1863579 568 0.TT.--;75.-.G 2.086 0.787 1-3 8531520 542 75.-.G;86.-.0 2.155 0.582 3006303 569 1.TA.--;88.G.- 2.086 0.537 cp n.) o 2296436 543 0.T.-;76.GG.-T 2.154 0.679 4236935 570 4.T.-;76.G.- 2.081 0.919 n.) o 4249048 544 4.T.-;86.-.0 2.142 0.675 12138801 571 2.A.-;89.-.A 2.080 1.115 c,.) o 10547068 545 15.-.T;87.-.G 2.140 0.857 12164760 572 2.A.-;89.-.T 2.080 0.316 un =
un index SEQ ID muts lindexed index SEQ
ID muts lindexed MI 95% CI
MI 95% CI
NO NO

10288787 573 17.-.T;86.-.0 2.080 0.927 4242379 600 4.T.-;77.GA.- 2.008 0.985 n.) o 2664128 574 0.T.-;2.A.C;77.-.0 2.079 0.379 2259846 601 0.T.-;76.G.-;78.A.0 2.005 0.640 n.) o iz..1 2663861 575 0.T.-;2.A.C;76.G.-;78.A.0 2.078 0.700 6462092 602 16.-.C;87.-.A 2.001 0.983 .6.
--.1 oe 2726063 576 0.T.-;2.A.C;78.A.T 2.078 0.972 4312495 603 4.T.-;73.AT.-G 1.997 0.708 oe n.) 4232837 577 4.T.-;76.GG.-C 2.069 0.580 2668714 604 0.T.-;2.A.C;81.GA.-C 1.996 0.678 3001194 578 1.TA.--;77.-.A 2.063 0.629 2294477 605 0.T.-;78.AG.-T 1.994 0.703 2048069 579 0.TT.-;2.A.G;76.G.- 2.059 1.413 12198135 606 2.A.-;77.-.T 1.994 1.433 2653681 580 2.A.C;0.T.-;75.-.A 2.052 0.427 4238150 607 4.T.-;77.-.A 1.993 0.762 2265126 581 0.T.-;88.G.- 2.050 0.557 3019738 608 1.TA.--;87.-.T 1.992 0.532 2739399 582 0.T.-;2.A.C;73.A.G 2.049 1.003 2352050 609 0.T.-;17.-.T 1.991 0.852 7250543 583 27.-.C;78.-.0 2.047 1.480 2705912 610 2.A.C;0.T.-;83.-.0 1.990 0.585 P
2747651 584 0.T.-;2.A.C;66.CT.- 2.047 0.900 6478822 611 16.-.C;74.-.T 1.989 0.477 ,..
, r., 12437734 585 1.TAC.--;78.A.- 2.043 0.615 2665913 612 2.A.C;0.T.-;79.GA.-C 1.987 1.186 ' .3 ,..
o 2826230 586 0.T.-;2.A.C;15.-.T 2.042 0.538 3331447 613 2.A.G;0.T.-;76.GG.-T 1.985 0.958 "
r., ,--µ, 2709008 587 2.A.C;0.T.-;82.A.-;84.A.T 2.037 1.246 3186538 614 2.A.G;0.T.-;27.-.A 1.983 1.530 , r., , 3005336 588 1.TA.--;86.-.A 2.034 0.483 2738784 615 2.A.C;0.T.-;73.AT.-G 1.977 0.623 .
4301274 589 4.T.-;76.G.-;78.A.T 2.028 0.873 7832272 616 55.-.G 1.977 0.882 3018865 590 1.TA.--;86.C.- 2.025 0.616 4297458 617 4.T.-;76.-.G 1.976 0.997 2699310 591 2.A.C;0.T.-;86.C.- 2.023 0.564 3334291 618 2.A.G;0.T.-;75.-.G 1.975 0.654 2279026 592 0.T.-;89.A.- 2.022 1.568 2212416 619 0.T.-;27.-.0 1.974 1.458 7248209 593 27.-.C;82.A.- 2.022 1.627 8752897 620 55.-.T;76.G.- 1.972 0.468 10562113 594 15.-.T;76.-.T 2.020 0.858 2293333 621 0.T.-;76.-.G 1.970 0.514 IV
n 7181373 595 27.-.A;76.G.- 2.014 1.908 7180386 622 27.-.A;76.GG.-A 1.969 1.667 1-3 10559019 596 15.-.T;76.-.G 2.014 0.753 2996180 623 1.TA.--;75.-.A 1.967 0.476 cp n.) o 3018452 597 1.TA.--;88.-.T 2.013 0.626 7238423 624 27.-.C;74.T.- 1.963 1.563 n.) o 12118457 598 2.A.-;76.-.A 2.011 1.170 2261752 625 0.T.-;77.GA.- 1.962 0.503 c,.) o 2805043 599 2.A.C;0.T.-;28.-.0 2.010 1.524 10282247 626 17.-.T;76.GG.-C 1.960 0.719 un =
un index SEQ ID muts lindexed index SEQ
ID muts lindexed MI 95% CI
MI 95% CI
NO NO

4230973 627 4.T.-;76.GG.-A 1.958 0.723 3003294 654 1.TA.--;77.GA.-- 1.896 0.506 n.) o 4276520 628 4.T.-;86.-.G 1.958 0.901 12121216 655 2.A.-;75.-.0 1.895 0.610 n.) o iz..1 2675193 629 0.T.-;2.A.C;88.GA.-C 1.957 0.878 2696635 656 0.T.-;2.A.C;89.AT.-G 1.894 0.882 .6.
--.1 oe 13101476 630 -1.GT.--;75.-.G 1.952 0.439 12130978 657 2.A.-;81.GA.-C 1.891 0.936 oe n.) 7203209 631 27.G.-;76.GG.-C 1.952 1.709 6475473 658 16.-.C;78.A.- 1.889 0.581 2724398 632 0.T.-;2.A.C;78.A.G 1.947 0.801 1853356 659 0.TT.--;76.G.- 1.885 0.802 10309365 633 17.-.T;78.-.T 1.947 1.542 8544082 660 75.-.G;87.-.G 1.884 0.536 10520418 634 15.-.T;74.T.- 1.945 0.728 2884429 661 1.-.C;76.G.- 1.884 0.673 10300394 635 17.-.T;87.-.G 1.944 1.037 6368955 662 17.-.A;76.-.G 1.882 0.843 4248302 636 4.T.-;88.G.- 1.937 0.857 2746170 663 .. 2.A.C;0.T.-;66.CT.-G .. 1.880 .. 0.517 7240856 637 27.-.C;76.G.-;78.A.0 1.937 1.188 4226314 664 4.T.-;74.-.0 1.874 0.901 P
4313003 638 4.T.-;73.A.G 1.935 0.688 6304607 665 16.-.A;76.G.- 1.873 0.523 ,..
, r., n.) 2467599 639 1.TA.--;3.C.A;76.GG.-T
1.923 1.105 2583788 666 0.T.-;2.A.C;27.G.- 1.873 1.388 ' .3 n.) ,..
1--, 2279202 640 0.T.-;89.-.T 1.921 0.709 2255694 667 0.T.-;76.-.A 1.869 0.837 "
r., ,--µ, 2259410 641 0.T.-;77.-.A 1.920 0.417 7249882 668 27.-.C;80.A.- 1.867 1.645 , r., , 4305674 642 4.T.-;75.-.G 1.915 1.089 10069481 669 19.-.T;75.-.0 1.864 0.645 .
6459602 643 16.-.C;76.G.- 1.915 0.642 2643173 670 0.T.-;2.A.C;70.T.- 1.864 1.689 2701869 644 0.T.-;2.A.C;86.-.G 1.914 0.477 12749699 671 0.-.T;75.-.G 1.863 0.757 2252978 645 0.T.-;74.-.G 1.911 0.602 7208859 672 27.G.-;87.-.G 1.862 1.687 6470049 646 16.-.C;87.-.G 1.910 0.715 4271233 673 4.T.-;89.-.0 1.854 0.839 12134362 647 2.A.-;86.-.A 1.907 0.661 6455215 674 16.-.C;73.-.A 1.850 0.825 12209524 648 2.A.-;73.A.0 1.901 1.154 2816525 675 0.T.-;2.A.C;19.-.T 1.848 0.369 IV
n 2260529 649 0.T.-;79.G.- 1.900 0.829 2292594 676 0.T.-;78.A.- 1.846 0.313 1-3 2690549 650 0.T.-;2.A.C;98.-.T 1.899 0.954 2287708 677 0.T.-;82.AA.-T 1.846 0.408 cp n.) o 10073100 651 19.-.T;88.G.- 1.898 0.782 2721779 678 2.A.C;0.T.-;78.A.- 1.842 0.677 n.) o 4239969 652 4.T.-;79.G.- 1.898 0.794 1945942 679 0.TT.--;2.A.C;75.-.G 1.842 1.271 c,.) o 3026047 653 1.TA.--;81.GA.-T 1.896 0.555 12111705 680 2.A.-;74.-.0 1.841 0.669 un =
un index SEQ ID muts lindexed index SEQ
ID muts lindexed MI 95% CI

NO NO

2567750 681 0.T.-;2.A.C;16.-.0 1.840 0.427 7180118 708 27.-.A;75.-.A 1.801 1.525 n.) o 2463364 682 1.TA.--;3.C.A;87.-.G 1.839 0.821 10081203 709 19.-.T;86.C.- 1.799 0.502 n.) o iz..1 3031594 683 1.TA.--;78.AG.-T 1.839 0.620 10532156 710 15.-.T;86.-.0 1.797 1.070 .6.
--.1 oe 10199376 684 18.-.G;75.-.G 1.837 1.238 2749667 711 2.A.C;0.T.-;65.GC.-T 1.795 0.642 oe n.) 4272444 685 4.T.-;89.A.- 1.837 0.998 12139228 712 2.A.-;90.-.0 1.794 1.201 9610551 686 28.-.C;78.A.- 1.836 1.802 10288547 713 17.-.T;88.G.- 1.794 1.193 2737747 687 0.T.-;2.A.C;73.A.0 1.833 1.293 4331367 714 4.T.-;55.-.T 1.793 0.481 12113430 688 2.A.-;74.-.G 1.828 0.753 2725463 715 2.A.C;0.T.-;78.-.T 1.792 0.507 10530413 689 15.-.T;85.TC.-G 1.825 1.155 2718857 716 0.T.-;2.A.C;79.GA.-T 1.792 0.900 12176759 690 2.A.-;83.-.T 1.824 1.046 2247247 717 0.T.-;72.-.A 1.792 0.887 12127185 691 2.A.-;79.G.- 1.824 0.606 12125011 718 2.A.-;77.-.A 1.786 0.527 P
4288099 692 4.T.-;81.GA.-T 1.824 0.753 4225246 719 4.T.-;74.T.- 1.786 0.629 ,..
, r., 12196850 693 2.A.-;78.A.T 1.821 1.086 12165722 720 2.A.-;88.-.T 1.786 1.273 ,..
n.) 6457366 694 16.-.C;75.-.A 1.821 0.638 2733129 721 0.T.-;2.A.C;75.C.- 1.786 0.561 "
r., ,--µ, 12105140 695 2.A.-;72.-.0 1.818 0.700 2469676 722 1.TA.--;3.C.A;73.A.- 1.785 1.174 , r., , 1944577 696 0.TT.--;2.A.C;78.A.- 1.817 1.170 3018172 723 1.TA.--;89.-.T 1.785 0.757 .
4293546 697 4.T.-;78.AG.-C 1.816 1.015 12196049 724 2.A.-;78.-.T 1.782 0.754 9996838 698 19.-.G;74.-.T 1.814 0.800 9612063 725 28.-.C;74.-.T 1.782 1.618 10301024 699 17.-.T;86.-.G 1.814 0.967 10547909 726 15.-.T;86.-.G 1.781 0.818 2308228 700 0.T.-;66.C.- 1.811 0.756 12194342 727 2.A.-;78.A.-;80.A.- 1.780 1.289 7835938 701 55.-.G;75.-.G 1.811 1.112 4228855 728 4.T.-;75.-.A 1.776 0.897 3005841 702 1.TA.--;87.-.A 1.811 0.806 10546613 729 15.-.T;86.C.- 1.776 0.859 IV
n 12169698 703 2.A.-;86.-.G 1.808 0.857 10547538 730 15.-.T;87.-.T 1.772 1.080 1-3 3028597 704 1.TA.--;78.AG.-C 1.803 0.743 10519772 731 15.-.T;73.-.A 1.771 0.624 cp n.) o 7191855 705 27.-.A;75.CG.-T 1.802 1.430 8510297 732 77.G.T 1.770 1.239 n.) o 9972503 706 19.-.G;74.T.- 1.802 0.750 12119606 733 2.A.-;76.GG.-C 1.768 1.110 c,.) o 4026979 707 3.-.C;75.-.G 1.802 1.374 2669299 734 0.T.-;2.A.C;85.TC.-A 1.767 0.842 un =
un index SEQ ID muts lindexed index SEQ
ID muts lindexed MI 95% CI
MI 95% CI
NO NO

6469807 735 16.-.C;86.C.- 1.765 0.759 4200182 762 4.T.-;55.-.G 1.721 1.233 n.) o 10197299 736 18.-.G;76.-.G 1.764 0.832 2281298 763 0.T.-;86.-.G 1.720 0.460 n.) o iz..1 3344225 737 2.A.G;0.T.-;73.A.- 1.762 1.216 7182097 764 27.-.A;77.GA.-- 1.719 1.318 .6.
--.1 oe 2456917 738 1.TA.--;3.C.A;75.-.A 1.761 1.203 2251662 765 0.T.-;74.T.- 1.719 0.428 oe n.) 10307233 739 17.-.T;78.AG.-C 1.760 1.101 1904870 766 0.TTA.---;3.C.A;76.G.- 1.715 1.345 12314352 740 2.A.-;15.-.T 1.758 0.436 10553996 767 15.-.T;81.GA.-T 1.715 0.963 12177388 741 2.A.-;82.AA.-- 1.751 0.615 10202590 768 18.-.G;73.A.- 1.715 0.822 2694455 742 0.T.-;2.A.C;91.A.-;93.A.G 1.751 1.015 3028839 769 1.TA.--;78.-.0 1.713 0.450 3040066 743 1.TA.--;73.A.- 1.750 0.690 3304552 770 0.T.-;2.A.G;89.-.T 1.713 0.767 10081633 744 19.-.T;87.-.T 1.750 0.917 4247308 771 4.T.-;87.-.A 1.711 0.766 4246508 745 4.T.-;86.-.A 1.749 0.939 4318521 772 4.T.-;66.CT.-G 1.710 0.957 P
4301580 746 4.T.-;77.-.T 1.744 0.701 7247759 773 27.-.C;86.-.G 1.710 1.198 , r., n.) A n.) 10181172 747 18.-.G;75.-. 1.743 1.016 10198320 774 18.-.G;76.GG.-T
1.709 0.701 ' .3 12200668 748 2.A.-;76.-.T 1.741 0.873 2457655 775 1.TA.--;3.C.A;76.GG.-C 1.709 1.260 "
r., , ' 10524336 749 15.-.T;76.GG.-C 1.738 0.390 3032520 776 1.TA.--;76.G.-;78.A.T 1.709 0.754 , r., , 3007212 750 1.TA.--;89.-.A 1.738 1.072 2702792 777 0.T.-;2.A.C;86.CC.-T 1.709 0.742 .
10526271 751 15.-.T;76.G.- 1.738 1.098 12171374 778 2.A.-;84.AT.-- 1.709 1.239 10561166 752 15.-.T;77.-.T 1.737 0.745 10192666 779 18.-.G;87.-.G 1.706 0.672 2663037 753 2.A.C;0.T.-;77.-.A 1.732 0.417 2642318 780 2.A.C;0.T.-;72.-.A 1.703 0.651 12136525 754 2.A.-;88.G.- 1.731 0.578 2718074 781 2.A.C;0.T.-;77.GA.--;82.A.T 1.700 1.191 8758832 755 55.-.T;78.A.- 1.731 0.641 12191670 782 2.A.-;78.A.- 1.697 0.819 1864295 756 0.TT.--;75.CG.-T 1.729 0.424 2456219 783 1.TA.--;3.C.A;74.T.- 1.696 1.260 1-0 n 10550736 757 15.-.T;82.A.-;84.A.G 1.728 0.888 2457365 784 1.TA.--;3.C.A;76.GG.-A 1.695 0.951 1-3 2657071 758 2.A.C;0.T.-;76.-.A 1.728 1.206 8538180 785 75.-.G 1.695 0.416 cp n.) o 2059338 759 0.TT.--;2.A.G;75.-.G 1.725 1.054 3020581 786 1.TA.--;86.CC.-T 1.693 1.160 n.) o 12182224 760 2.A.-;82.AA.-T 1.722 0.599 10281916 787 17.-.T;76.-.A 1.693 0.649 -1 o 2671130 761 2.A.C;0.T.-;85.TC.-G 1.721 0.884 2707684 788 0.T.-;2.A.C;82.A.-;84.A.G 1.692 1.346 un =
un index SEQ ID muts lindexed index SEQ
ID muts lindexed MI 95% CI
MI 95% CI
NO NO

2676761 789 0.T.-;2.A.C;90.-.G 1.689 1.000 2297191 815 0.T.-;76.-.T 1.652 0.458 n.) o 7213979 790 27.G.-;75.CG.-T 1.689 1.195 2126158 816 0.TTA.---;3.C.G;87.-.G 1.650 1.318 n.) o iz..1 2459101 791 1.TA.--;3.C.A;77.GA.-- 1.687 0.967 2283617 817 0.T.-;83.-.0 1.649 1.421 .6.
--.1 oe 8123571 792 75.-.C;86.-.0 1.686 0.454 2654520 818 2.A.C;0.T.-;75.CG.-A 1.647 0.574 oe n.) 12207287 793 2.A.-;75.CG.-T 1.685 0.564 3332543 819 0.T.-;2.A.G;76.-.T 1.645 0.844 2740245 794 2.A.C;0.T.-;70.-.T 1.685 1.013 9604425 820 28.-.C;88.G.- 1.644 1.218 10531744 795 15.-.T;88.G.- 1.685 1.172 12109255 821 2.A.-;73.-.A 1.644 0.930 2669798 796 2.A.C;0.T.-;82.-.A 1.684 0.486 12438229 822 1.TAC.---;76.GG.-T 1.642 0.689 2294771 797 0.T.-;78.-.T 1.684 0.366 8153054 823 77.G.0 1.641 1.385 7213033 798 27.G.-;76.GG.-T 1.682 1.554 10308482 824 17.-.T;76.-.G 1.641 1.127 7829581 799 55.-.G;76.G.- 1.682 1.158 10300026 825 17.-.T;86.C.- 1.641 1.228 P
.
2808092 800 0.T.-;2.A.C;28.-.T 1.680 1.571 2715234 826 2.A.C;0.T.-;80.AG.-C 1.640 1.476 ,..
, N) n.) A
n.) 2960043 801 1.T.--;27.-.0 1.676 1.353 10532541 827 15.-.T;90.T.- 1.640 1.020 ,..
.6.
10506564 802 15.-.T;55.-.G 1.675 1.443 12721860 828 0.-.T;76.G.- 1.640 0.367 N) N) ,--µ, 4315349 803 4.T.-;73.A.T 1.668 0.705 2460008 829 1.TA.--;3.C.A;86.-.0 1.639 0.936 , N) , 2705067 804 2.A.C;0.T.-;82.A.- 1.668 0.498 2264044 830 0.T.-;86.-.A 1.639 0.512 .
3330280 805 0.T.-;2.A.G;76.G.-;78.A.T 1.667 0.948 12188811 831 2.A.-;78.AG.-C 1.638 0.776 9630969 806 16. 12432569 832 1.TAC.---;76.GG.-A 1.637 0.883 1.665 1.315 .CTCATTACTTTG;75.-.A
9602947 833 28.-.C;75.-.0 1.636 1.558 12173513 807 2.A.-;82.A.- 1.664 0.734 2994003 834 1.TA.--;74.T.- 1.634 0.542 3280346 808 0.T.-;2.A.G;87.-.A 1.663 1.204 12213405 835 2.A.-;73.A.- 1.634 0.736 7238549 809 27.-.C;74.-.0 1.661 1.215 2719575 836 0.T.-;2.A.C;78.AG.-C 1.633 0.446 IV
8154695 810 76.G.-;78.A.0 1.661 0.368 n 2123173 837 0.TTA.---;3.C.G;76.G.- 1.632 1.511 1-3 10516784 811 15.-.T;72.-.A 1.660 0.597 10086342 838 19.-.T;78.-.0 1.631 0.477 cp n.) 10307953 812 17.-.T;78.A.- 1.660 0.824 12236371 839 2.A.-;55.-.T 1.630 0.850 2 o 12432835 813 1.TAC.---;75.-.0 1.654 0.814 -1 6473588 840 16.-.C;81.GA.-T 1.628 0.398 c,.) 12193344 814 2.A.-;76.-.G
1.654 0.664 o un 7240999 841 27.-.C;79.G.- 1.628 1.310 =
un index SEQ ID muts lindexed index SEQ
ID muts lindexed MI 95% CI
MI 95% CI
NO NO

12189370 842 2.A.-;78.-.0 1.625 0.715 3439310 869 0.T.-;2.A.G;15.-.T 1.589 0.341 n.) o 3005003 843 1.TA.--;85.TC.-G 1.625 0.820 2718364 870 0.T.-;2.A.C;80.A.T 1.588 1.149 n.) o iz..1 10185851 844 18.-.G;86.-.0 1.622 0.720 4223967 871 4.T.-;73.-.A 1.587 0.646 .6.
--.1 oe 2725020 845 0.T.-;2.A.C;78.AG.-T 1.622 0.696 4271617 872 4.T.-;89.AT.-G 1.587 1.233 oe n.) 12212274 846 2.A.-;70.-.T 1.621 1.038 10460510 873 16.C.-;76.GG.-A 1.587 0.788 8470264 847 78.-.0 1.617 0.272 4227764 874 4.T.-;74.-.G 1.586 0.680 2286841 848 0.T.-;82.AA.-G 1.617 0.606 9994855 875 19.-.G;76.GG.-T 1.585 0.779 7241506 849 27.-.C;81.GA.-C 1.617 1.112 3272821 876 2.A.G;0.T.-;76.G.-;78.A.0 1.583 0.912 12163987 850 2.A.-;89.A.G 1.617 0.718 12110798 877 2.A.-;74.T.- 1.582 0.659 3364655 851 0.T.-;2.A.G;55.-.T 1.615 1.131 1975319 878 0.T.C;76.G.- 1.581 0.610 1904677 852 0.TTA.---;3.C.A;75.-.0 1.614 0.965 10316332 879 17.-.T;73.A.- 1.581 0.902 P
2712438 853 2.A.C;0.T.-;82.-.T 1.612 0.769 2720616 880 0.T.-;2.A.C;78.A.0 1.581 0.565 ,..
, r., n.) 14645004 854 -29.A.C;O.T.-;2.A.C;76.G.-1.610 0.433 8753785 881 55.-.T;86.-.0 1.581 0.908 ' .3 n.) ,..
un 10322550 855 17.-.T;55.-.T 1.608 0.835 8112378 882 76.-.A 1.580 0.965 "
r., ,--µ, 10304965 856 17.-.T;82.AA.-T 1.606 1.006 2819005 883 0.T.-;2.A.C;18.-.G 1.579 0.491 , r., , 10279228 857 17.-.T;74.-.0 1.603 0.965 8357828 884 87.-.G 1.579 0.261 .
3263089 858 2.A.G;0.T.-;74.-.G 1.603 0.944 6477023 885 16.-.C;76.GG.-T 1.577 0.802 2282393 859 0.T.-;82.A.-;85.T.G 1.602 1.047 12737747 886 0.-.T;87.-.G 1.577 0.587 2463251 860 1.TA.--;3.C.A;86.C.- 1.598 0.959 12309294 887 2.A.-;17.-.T 1.576 0.644 2459897 861 1.TA.--;3.C.A;88.G.- 1.596 0.725 2252133 888 0.T.-;74.-.0 1.576 0.340 1852430 862 0.TT.--;76.GG.-A 1.596 0.848 10567192 889 15.-.T;73.AT.-G 1.575 0.657 10305251 863 17.-.T;81.GA.-T 1.593 1.079 3261438 890 2.A.G;0.T.-;74.-.0 1.575 0.783 IV
n 9603994 864 28.-.C;85.TC.-A 1.593 1.339 15169229 891 -29.A.G;75.-.G 1.574 0.382 1-3 4319798 865 4.T.-;66.CT.-- 1.593 0.719 6128804 892 14.-.A;76.GG.-T 1.574 0.980 cp n.) o 3042484 866 1.TA.--;66.CT.-G 1.592 0.578 12197720 893 2.A.-;76.G.-;78.A.T 1.573 0.893 n.) o 8544184 867 75.-.G;87.-.T 1.592 0.631 3326919 894 2.A.G;0.T.-;76.-.G 1.573 0.783 c,.) o 2709867 868 2.A.C;0.T.-;82.AA.-C 1.590 0.506 12164376 895 2.A.-;89.A.- 1.572 1.400 un =
un index SEQ ID muts lindexed index SEQ
ID muts lindexed MI 95% CI

NO NO

2990209 896 1.TA.--;70.T.- 1.571 1.474 3416823 923 0.T.-;2.A.G;28.-.0 1.539 1.436 n.) o 8538220 897 75.-.G;132.G.T 1.571 0.465 9976094 924 19.-.G;76.G.- 1.539 0.749 n.) o iz..1 10068467 898 19.-.T;76.GG.-A 1.570 0.904 1852751 925 0.TT.--;76.GG.-C 1.537 0.770 .6.
--.1 oe 9697533 899 28.-.T;75.CG.-T 1.569 1.330 4314686 926 4.T.-;73.A.- 1.536 1.014 oe n.) 2958993 900 1.TA.--;27.-.A 1.568 1.255 6470272 927 16.-.C;87.-.T 1.536 0.597 3001629 901 1.TA.--;76.G.-;78.A.0 1.566 0.524 2673006 928 0.T.-;2.A.C;87.C.A 1.535 0.804 4291732 902 4.T.-;77.GA.--;82.A.T 1.565 1.310 12137377 929 2.A.-;86.-.0 1.535 0.546 4238868 903 4.T.-;76.G.-;78.A.0 1.564 0.830 12184036 930 2.A.-;80.AG.-C 1.532 1.352 3306461 904 0.T.-;2.A.G;87.-.G 1.564 0.717 10285242 931 17.-.T;77.-.0 1.530 1.164 1937976 905 2.A.C;0.TT.--;76.G.- 1.560 1.463 2263017 932 0.T.-;82.-.A 1.530 0.468 4172716 906 4.T.-;27.-.0 1.558 1.388 12163286 933 2.A.-;89.AT.-G 1.529 1.001 P
12185288 907 2.A.-;80.A.- 1.557 0.706 2706481 934 2.A.C;0.T.-;82.A.-;84.A.0 1.528 1.209 ,..
, r., n.) A 14813579 908 -29..C;75.-.G
1.557 0.415 4320578 935 4.T.-;66.C.- 1.527 0.995 ' n.) .3 o ,..
2468675 909 1.TA.--;3.C.A;75.CG.-T 1.553 0.931 3004121 936 1.TA.--;85.TC.-A 1.526 0.698 "
r., , ' 12195510 910 2.A.-;78.AG.-T 1.550 0.887 3269260 937 2.A.G;0.T.-;75.-.0 1.522 0.739 , r., , 4285997 911 4.T.-;82.AA.-G 1.549 0.782 7835518 938 55.-.G;76.-.G 1.519 0.935 .
3275841 912 2.A.G;0.T.-;77.GA.-- 1.549 0.526 10195401 939 18.-.G;81.GA.-T 1.519 0.776 3018032 913 1.TA.--;89.A.- 1.549 1.114 6477333 940 16.-.C;76.-.T 1.516 0.627 2301817 914 0.T.-;73.A.0 1.549 0.917 4171307 941 4.T.-;27.-.A 1.514 1.234 3305057 915 0.T.-;2.A.G;88.-.T 1.548 0.420 10299590 942 17.-.T;88.-.T 1.513 1.296 2122618 916 0.TTA.---;3.C.G;76.GG.-A 1.548 1.094 6478447 943 16.-.C;75.C.- 1.512 0.508 2289325 917 0.T.-;80.A.- 1.547 0.393 4249490 944 4.T.-;88.GA.-C 1.512 0.737 1-0 n 4291562 918 4.T.-;80.AG.-T 1.547 1.017 12220656 945 2.A.-;66.C.- 1.512 1.055 1-3 10557226 919 15.-.T;78.-.0 1.545 0.975 7240739 946 27.-.C;77.-.A 1.512 1.178 cp n.) o 12748115 920 0.-.T;76.GG.-T 1.545 0.710 10315246 947 17.-.T;73.AT.-G 1.511 1.010 n.) o 3026518 921 1.TA.--;80.AG.-C 1.544 1.241 1944754 948 0.TT.--;2.A.C;76.-.G 1.511 1.156 -1 o 10545028 922 15.-.T;89.-.0 1.542 0.579 3337255 949 2.A.G;0.T.-;74.-.T 1.510 0.678 un =
un index SEQ ID muts lindexed index SEQ
ID muts lindexed MI 95% CI
MI 95% CI
NO NO

6362999 950 17.-.A;76.G.- 1.509 1.043 4287312 977 4.T.-;82.AA.-T 1.473 0.577 n.) o 3017407 951 1.TA.--;89.-.0 1.509 0.465 3339492 978 2.A.G;0.T.-;73.AT.-C 1.472 1.445 n.) o iz..1 9973601 952 19.-.G;75.-.A 1.503 0.894 4290113 979 4.T.-;80.A.- 1.470 0.639 .6.
--.1 oe 12186826 953 2.A.-;80.AG.-T 1.501 0.813 2293835 980 0.T.-;78.A.-;80.A.- 1.469 0.867 wc'e 3035711 954 1.TA.--;75.C.- 1.500 0.592 6455860 981 16.-.C;74.-.0 1.468 0.527 8526584 955 76.-.T 1.499 0.320 2706303 982 0.T.-;2.A.C;82.AA.--;85.T.0 1.467 1.023 2211100 956 0.T.-;27.-.A 1.499 1.300 7252350 983 27.-.C;76.-.T 1.467 1.180 8558515 957 74.-.T 1.499 0.244 3277392 984 0.T.-;2.A.G;85.TC.-A 1.467 1.201 4321895 958 4.T.-;65.GC.-T 1.498 0.661 8538161 985 75.-.G;132.G.0 1.467 0.428 12204638 959 2.A.-;75.C.- 1.496 0.655 8202442 986 87.-.A 1.465 0.819 8118238 960 76.GG.-C 1.495 0.555 2898633 987 1.-.C;78.-.0 1.464 0.456 P
2348592 961 0.T.-;19.-.T 1.493 0.463 2648767 988 2.A.C;0.T.-;73.-.A 1.463 0.659 ,..
, r., n.) 3282394 962 0.T.-;2.A.G;88.GA.-C
1.491 1.144 6115163 989 14.-.A;88.G.- 1.463 0.529 ' .3 n.) ,..
--.1 9974216 963 19.-.G;76.GG.-A 1.490 0.650 10576534 990 15.-.T;55.-.T 1.461 0.556 "
r., , 3435006 964 0.T.-;2.A.G;17.-.T 1.488 0.572 1904556 991 0.TTA.---;3.C.A;76.GG.-C 1.461 1.089 ' , r., , 2291281 965 0.T.-;78.AG.-C 1.486 0.722 8073267 992 74.-.0 1.459 0.430 3013663 966 1.TA.--;99.-.G 1.484 0.730 8755280 993 55.-.T 1.458 0.638 7255023 967 27.-.C;70.-.T 1.484 1.384 2341059 994 0.T.-;28.-.0 1.457 1.284 4307384 968 4.T.-;75.C.- 1.483 0.592 3007006 995 1.TA.--;90.T.- 1.456 1.125 2702279 969 0.T.-;2.A.C;86.CC.-G 1.482 1.155 7833962 996 55.-.G;87.-.G 1.456 0.883 3036396 970 1.TA.--;74.-.T 1.480 0.455 4299868 997 4.T.-;78.-.T 1.456 0.940 10196645 971 18.-.G;78.-.0 1.479 0.758 8342692 998 89.A.G 1.455 0.975 1-0 n 4308690 972 4.T.-;74.-.T 1.479 0.955 2262741 999 0.T.-;85.TC.-A 1.451 0.583 1-3 4298804 973 4.T.-;78.A.G 1.477 0.725 1942088 1000 0.TT.--;2.A.C;86.C.- 1.450 1.216 cp n.) o 12125860 974 2.A.-;76.G.-;78.A.0 1.476 0.782 10200245 1001 18.-.G;74.-.T 1.448 0.938 n.) o 2675530 975 0.T.-;2.A.C;90.T.- 1.474 1.266 4219211 1002 4.T.-;72.-.A 1.447 0.549 c,.) o 7242260 976 27.-.C;88.G.- 1.473 1.439 2457931 1003 1.TA.--;3.C.A;75.-.0 1.444 0.736 un =
un index SEQ ID muts lindexed index SEQ
ID muts lindexed MI 95% CI

NO NO

3038631 1004 1.TA.--;73.AT.-G 1.444 0.560 2710592 1031 2.A.C;0.T.-;81.-.G 1.420 0.684 n.) o 12753950 1005 0.-.T;73.A.- 1.444 0.573 8537382 1032 75.-.G;121.C.A 1.419 0.408 n.) o iz..1 2129014 1006 0.TTA.---;3.C.G;75.-.G 1.440 1.366 12434064 1033 1.TAC.---;86.-.0 1.417 0.739 .6.
--.1 oe 7833901 1007 55.-.G;86.C.- 1.439 0.671 12438652 1034 1.TAC.---;75.C.- 1.417 0.894 wc'e 10066878 1008 19.-.T;74.-.0 1.439 0.663 8105679 1035 76.GG.-A 1.416 0.238 2714726 1009 0.T.-;2.A.C;77.GA.--;83.A.T 1.439 0.739 8089861 1036 75.-.A;86.-.0 1.414 0.397 12106738 1010 2.A.-;72.-.G 1.438 1.201 10177945 1037 18.-.G;72.-.A 1.414 0.836 2720418 1011 0.T.-;2.A.C;77.GA.--;80.A.0 1.436 1.201 4243445 1038 4.T.-;81.GA.-C 1.413 0.887 2291924 1012 0.T.-;78.A.0 1.436 0.937 8123491 1039 75.-.C;88.G.- 1.412 0.441 9991025 1013 19.-.G;81.GA.-T 1.434 0.688 4313666 1040 4.T.-;70.-.T 1.411 0.506 4243954 1014 4.T.-;85.TC.-A 1.433 0.674 7180551 1041 27.-.A;76.-.A 1.410 1.181 P
6362816 1015 17.-.A;75.-.0 1.433 0.887 6534510 1042 17.-.G;76.GG.-T 1.407 0.941 ,..
, r., n.) 8204227 1016 87.C.A 1.432 1.065 3025550 1043 1.TA.--;82.AA.-T 1.407 0.570 n.) ,..
oe 1980019 1017 0.T.C;78.A.- 1.431 0.702 10275000 1044 17.-.T;71.-.0 1.406 0.754 "
r., , 8142815 1018 76.G.-;130.T.G 1.429 0.271 8530347 1045 75.-C.GA 1.406 0.333 ' , r., , 10554966 1019 15.-.T;80.A.- 1.429 1.003 12438782 1046 1.TAC.---;74.-.T 1.404 0.868 2702620 1020 0.T.-;2.A.C;86.C.T 1.427 0.892 2724111 1047 2.A.C;0.T.-;78.A.-;80.A.- 1.403 1.013 8142856 1021 76.G.-;132.G.0 1.427 0.238 12682492 1048 0.-.T;27.-.0 1.402 1.266 12012995 1022 2.A.-;16.-.0 1.425 0.515 8336449 1049 89.-.0 1.400 0.251 4284095 1023 4.T.-;82.AA.-C 1.424 0.718 2994450 1050 1.TA.--;74.-.0 1.399 0.436 10546168 1024 15.-.T;88.-.T 1.424 1.002 10070026 1051 19.-.T;76.G.- 1.399 0.599 8128579 1025 75.-.0 1.424 0.273 4246898 1052 4.T.-;86.CC.-A 1.398 0.996 1-0 n 2703946 1026 2.A.C;0.T.-;82.A.-;85.T.G 1.423 1.276 2056199 1053 0.TT.--;2.A.G;82.AA.-T 1.398 1.059 1-12433040 1027 1.TAC.---;76.G.- 1.423 0.852 2726405 1054 0.T.-;2.A.C;77.G.T 1.398 0.989 cp n.) o 12162901 1028 2.A.-;89.-.0 1.422 0.831 8093322 1055 75.-.A 1.396 0.309 n.) o 2814556 1029 0.T.-;2.A.C;19.-.G 1.420 0.572 4239175 1056 4.T.-;77.-.0 1.396 0.979 c,.) o 8142933 1030 76.G.-;132.G.T 1.420 0.297 3031832 1057 1.TA.--;78.-.T 1.395 0.529 un =
un index SEQ ID muts lindexed index SEQ
ID muts lindexed MI 95% CI

NO NO

2303944 1058 0.T.-;73.A.- 1.395 0.686 2672282 1085 2.A.C;0.T.-;86.CC.-A 1.376 0.805 n.) o 2255406 1059 0.T.-;76.GG.-- 1.395 1.055 14798941 1086 -29.A.C;75.-.0 1.376 0.255 n.) o 2468522 1060 1.TA.--;3.C.A;74.-.T 1.394 0.748 12031760 1087 2.A.-;27.G.- 1.375 1.375 .6.

oe 8543995 1061 75.-.G;86.C.- 1.393 0.372 2201185 1088 0.T.-;16.-.0 1.373 0.446 wc'e 8348831 1062 88.-.T 1.392 0.333 2400173 1089 1.-.A;76.G.- 1.372 0.596 2899043 1063 1.-.C;78.A.- 1.392 0.693 10088256 1090 19.-.T;76.G.-;78.A.T 1.370 0.715 6611143 1064 18.C.-;75.-.A 1.392 0.602 10284913 1091 17.-.T;77.-.A 1.370 1.090 8142880 1065 76.G.- 1.391 0.256 10545701 1092 15.-.T;89.A.- 1.370 1.003 4294538 1066 4.T.-;78.A.0 1.390 0.607 8212851 1093 86.-.0 1.369 0.540 447196 1067 -27.C.A;75.-.G 1.390 0.365 8132895 1094 75.-.C;86.C.- 1.368 0.297 3338210 1068 2.A.G;0.T.-;75.CG.-T 1.390 0.686 3281950 1095 2.A.G;0.T.-;86.-.0 1.368 0.907 P
8538250 1069 75.-.G;131.A.0 1.389 0.442 1858655 1096 0.TT.--;87.-.G 1.368 0.620 ,..
, r., n.) 10302419 1070 17.-.T;83.-.0 1.388 1.345 12737396 1097 0.-.T;86.C.- 1.365 0.552 ' .3 n.) ,..
o 3169133 1071 0.T.-;2.A.G;16.-.0 1.388 0.627 6474033 1098 16.-.C;80.A.- 1.363 0.562 "
r., , 1855234 1072 0.TT.--;86.-.0 1.387 0.590 2646406 1099 0.T.-;2.A.C;72.-.G 1.363 1.115 ' , r., , 3027053 1073 1.TA.--;80.A.- 1.386 0.444 3020097 1100 1.TA.--;86.-.G 1.363 0.580 8142905 1074 76.G.-;133.A.0 1.386 0.312 12160739 1101 2.A.-;91.A.-;93.A.G 1.363 1.067 2465375 1075 1.TA.--;3.C.A;81.GA.-T 1.386 0.850 14919005 1102 -29.A.C;2.A.-;76.G.- 1.362 0.433 8137397 1076 76.G.-;98.-.A 1.385 0.658 10527714 1103 15.-.T;79.G.- 1.362 0.847 3304306 1077 2.A.G;0.T.-;89.A.- 1.384 1.226 3023033 1104 1.TA.--;82.A.-;84.A.G 1.361 1.195 8537231 1078 75.-.G;120.C.A 1.383 0.451 2467773 1105 1.TA.--;3.C.A;76.-.T 1.361 0.680 4299393 1079 4.T.-;78.AG.-T 1.382 1.034 2284824 1106 0.T.-;83.-.T 1.361 0.848 1-0 n 3295454 1080 2.A.G;0.T.-;99.-.G 1.382 1.039 9987305 1107 19.-.G;87.-.G 1.360 0.734 1-3 8519489 1081 76.GG.-T 1.380 0.164 2628450 1108 2.A.C;0.T.-;65.GC.-A 1.360 0.861 cp n.) o 3264318 1082 2.A.G;0.T.-;75.-.A 1.379 0.703 8531228 1109 75.-.G;87.-.A 1.360 0.691 n.) o -a 5 3266116 1083 2.A.G;0.T.-;76.GG.-A 1.379 0.672 1939243 1110 0.TT.--;2.A.C;86.-.0 1.358 0.943 c,.) o 2997992 1084 1.TA.--;76.-.A 1.378 0.700 3050495 1111 1.TA.--;55.-.T 1.358 0.880 un =
un index SEQ ID muts lindexed index SEQ
ID muts lindexed MI 95% CI
MI 95% CI
NO NO

7835450 1112 55.-.G;78.A.- 1.358 0.698 6313836 1138 16.-.A;78.A.- 1.342 0.715 n.) o 12702721 1113 0.-.T;55.-.G 1.357 0.531 6455586 1139 16.-.C;74.T.- 1.341 0.589 n.) o iz..1 4231994 1114 4.T.-;76.-.A 1.357 0.799 10069022 1140 19.-.T;76.GG.-C 1.339 0.689 .6.
--.1 oe 10185683 1115 18.-.G;88.G.- 1.357 1.038 8538125 1141 75.-.G;130.T.G 1.339 0.405 wc'e 2709497 1116 2.A.C;0.T.-;82.A.0 1.356 1.204 8208034 1142 88.G.- 1.339 0.227 8330844 1117 91.A.G 1.355 1.033 4210228 1143 4.T.-;65.G.- 1.338 0.726 10287644 1118 17.-.T;85.TC.-G 1.355 1.182 8555144 1144 74.-.T;86.-.0 1.336 0.495 9976346 1119 19.-.G;77.-.A 1.355 0.744 2211631 1145 0.T.-;27.G.- 1.336 1.023 8759277 1120 55.-.T;75.-.G 1.353 0.800 14799468 1146 -29.A.C;76.G.- 1.335 0.265 2711676 1121 2.A.C;0.T.-;82.AA.-G 1.352 0.772 3023524 1147 1.TA.--;82.AA.-- 1.335 0.777 10199887 1122 18.-.G;75.C.- 1.351 0.818 14921453 1148 -29.A.C;2.A.-;75.-.G 1.334 0.448 P
.
12131652 1123 2.A.-;85.TC.-A 1.351 1.139 2465666 1149 1.TA.--;3.C.A;80.A.- 1.334 1.225 ,..
, N) n.) A 8628479 1124 66.CT.-G;76.G.-1.351 0.362 2124272 1150 0.TT.---;3.C.G;86.-.0 1.333 1.021 ' .3 ,..
o 2459762 1125 1.TA.--;3.C.A;87.-.A 1.350 1.009 4366553 1151 4.T.-;28.-.0 1.333 1.147 "
.
N) , 8647329 1126 66.C.T 1.350 1.188 15160651 1152 -29.A.G;75.-.0 1.333 0.280 ' , N) , 6526262 1127 17.-.G;76.G.- 1.350 1.265 2248937 1153 0.T.-;70.T.-;73.A.0 1.329 1.289 ' 2279498 1128 0.T.-;88.-.T 1.350 0.488 10307622 1154 17.-.T;78.A.0 1.329 0.893 2719218 1129 0.T.-2670634 1155 0.T.-;2.A.C;85.TC.-- 1.327 0.861 ;2.A.C;79.GAGAAA.TTTCT 1.349 1.087 10180147 1156 18.-.G;74.-.0 1.326 0.933 C
10288203 1157 17.-.T;87.-.A 1.325 0.741 1858516 1130 0.TT.--;86.C.- 1.349 1.337 14806896 1158 -29.A.C;87.-.G 1.324 0.256 14798574 1131 -29.A.C;76.GG.-C 1.347 0.500 2708627 1159 0.T.-;2.A.C;82.AA.-- 1.323 0.576 1-0 10178596 1132 18.-.G;72.-.0 1.346 0.766 n 3260655 1160 2.A.G;0.T.-;74.T.- 1.322 0.641 1-3 8118222 1133 76.GG.-C;132.G.0 1.346 0.517 12719454 1161 0.-.T;76.GG.-A 1.322 0.483 cp 12181387 1134 2.A.-;82.-.T
1.345 0.639 n.) o 12432022 1162 1.TAC.---;74.-.0 1.321 0.647 n.) 10285141 1135 17.-.T;76.G.-;78.A.0 1.345 0.980 4245923 1163 4.T.-;85.TC.-G 1.321 1.255 c,.) 8565359 1136 75.CG.-T 1.345 0.288 o 8363261 1164 87.-.T 1.321 0.482 un =
8142963 1137 76.G.-;131.A.0 1.344 0.259 un index SEQ ID muts lindexed index SEQ
ID muts lindexed MI 95% CI
MI 95% CI
NO NO

2128723 1165 0.TTA.---;3.C.G;76.GG.-T 1.318 1.199 8757116 1191 55.-.T;87.-.G 1.293 0.601 n.) o 8514493 1166 77.-.T 1.318 0.804 2701481 1192 0.T.-;2.A.C;87.C.T 1.292 0.555 n.) o iz..1 3330625 1167 0.T.-;2.A.G;77.-.T 1.317 1.252 6458094 1193 16.-.C;76.GG.-A 1.290 1.072 .6.
--.1 oe 10279842 1168 17.-.T;74.-.G 1.316 0.997 8096141 1194 75.-.A;87.-.G 1.289 0.400 2 3271300 1169 2.A.G;0.T.-;76.G.- 1.315 0.602 1937383 1195 0.TT.--;2.A.C;76.GG.-C 1.288 1.058 12209957 1170 2.A.-;73.-.G 1.314 1.123 10527226 1196 15.-.T;76.G.-;78.A.0 1.288 0.941 2295677 1171 0.T.-;76.G.-;78.A.T 1.314 0.644 2461285 1197 1.TA.--;3.C.A 1.288 1.104 7188615 1172 27.- 1.312 1.251 9999142 1198 19.-.G;73.A.- 1.286 0.905 .A;79.GAGAAA.TTTCTC
8190839 1199 85.TC.-- 1.286 0.969 8638657 1173 66.CT.-G;78.A.- 1.311 0.331 4021093 1200 3.-.C;87.-.G 1.285 0.949 6470437 1174 16.-.C;86.-.G 1.310 0.430 8128562 1201 75.-.C;132.G.0 1.284 0.296 P
12102732 1175 2.A.-;72.-.A
1.307 0.918 0 4026117 1202 3.-.C;76.GG.-T 1.282 0.871 ,..
, 8142718 1176 76.G.-;129.C.A
1.305 0.257 .
"
n.) A 3458694 1203 0.TTC.----;75.-.0 1.282 1.236 ' .3 ,..
1--, 8156448 1177 77.-.0 1.304 0.590 2402393 1204 1.-.A;87.-.A 1.282 0.828 "
.
1852995 1178 0.TT.--;75.-.0 1.303 0.901 r., , 1852100 1205 0.TT.--;75.-.A 1.281 0.682 ' , 2887175 1179 1.-.C;88.G.-1.303 0.598 r., , 3325688 1206 2.A.G;0.T.-;78.A.- 1.281 0.892 ' 2263396 1180 0.T.-;85.T.- 1.302 1.134 2742029 1207 0.T.-;2.A.C;73.A.T 1.281 0.548 1825818 1181 0.TT.-A;76.G.- 1.302 1.110 6577492 1208 18.-.A;86.-.0 1.280 0.718 8344169 1182 89.A.- 1.302 1.226 12218636 1209 2.A.-;66.CT.-G 1.279 0.773 2709285 1183 2.A.C;0.T.-;82.-.0 1.301 0.894 8219007 1210 89.-.A 1.279 1.111 3023675 1184 1.TA.--;82.A.-;84.A.T 1.300 0.818 6369323 1211 17.-.A;76.GG.-T 1.278 0.804 10084841 1185 19.-.T;81.GA.-T 1.298 0.600 2651674 1212 0.T.-;2.A.C;74.TC.-- 1.278 1.277 1976248 1186 0.T.C;86.-.0 1.298 0.826 n 12717259 1213 0.-.T;74.-.0 1.277 0.541 1-3 12154344 1187 2.A.-;99.-.G 1.296 1.001 15160113 1214 -29.A.G;76.GG.-A
1.277 0.270 cp n.) 13097626 1188 -1.GT.--;76.G.- 1.295 0.442 2900998 1215 1.-.C;76.-.T 1.277 0.460 2 o 6458438 1189 16.-.C;76.-.A
1.295 0.847 -1 1864123 1216 0.TT.--;74.-.T 1.275 0.783 c,.) 8150274 1190 77.-.A 1.294 0.229 o un 1936243 1217 0.TT.--;2.A.C;73.-.A 1.269 0.978 =
un index SEQ ID muts lindexed index SEQ
ID muts lindexed MI 95% CI
MI 95% CI
NO NO

10087310 1218 19.-.T;76.-.G 1.269 1.013 8538003 1245 75.-.G;128.T.G 1.255 0.362 n.) o 8128641 1219 131.A.C;75.-.0 1.268 0.347 8531397 1246 75.-.G;88.G.- 1.254 0.477 n.) o iz..1 2466267 1220 1.TA.--;3.C.A;78.-.0 1.268 0.761 10088571 1247 19.-.T;76.GG.-T 1.254 0.431 .6.
--.1 oe 14814370 1221 -29.A.C;74.-.T 1.268 0.225 10090672 1248 19.-.T;74.-.T 1.254 0.833 wc'e 8367586 1222 86.-.G 1.268 0.167 9978638 1249 19.-.G;87.-.A 1.254 0.821 14814654 1223 -29.A.C;75.CG.-T 1.267 0.300 10183679 1250 18.-.G;76.G.-;78.A.0 1.253 0.445 7178892 1224 27.-.A;72.-.0 1.267 1.242 2283016 1251 0.T.-;82.A.- 1.253 0.466 2713900 1225 0.T.-;2.A.C;82.AA.--;84.A.T 1.267 1.065 2695201 1252 0.T.-;2.A.C;91.A.G 1.253 0.804 12745658 1226 0.-.T;78.A.- 1.266 0.629 6475853 1253 16.-.C;76.-.G 1.251 0.663 12436108 1227 1.TAC.---;86.C.- 1.265 0.683 6111106 1254 14.-.A;76.GG.-A 1.250 0.738 8490474 1228 76, G;131.A.0 1.265 0.316 3082312 1255 1.TA.--;17.-.T 1.249 0.812 P
6479094 1229 16, C;75.CG.-T 1.264 0.658 10566255 1256 15.-.T;73.AT.-C 1.249 0.813 ,..
, r., n.) A 10280354 1230 17.-.T;75.-.
1.264 1.255 10070730 1257 19.-.T;79.G.- 1.249 0.602 ' .3 ,..
n.) 10528666 1231 15.-.T;77.GA.- 1.264 1.070 14812876 1258 -29.A.C;76.GG.-T 1.248 0.151 "
r., , 10303386 1232 17.-.T;82.AA.- 1.264 1.142 1246999 1259 -15.T.G;76.G.- 1.247 0.225 ' , r., , 2355406 1233 0.T.-;15.-.T 1.262 0.700 8558498 1260 74.-.T;132.G.0 1.246 0.249 3032160 1234 1.TA.--;78.A.T 1.262 0.662 10518792 1261 15.-.T;72.-.G
1.246 0.489 7237755 1235 27.-.C;72.-.0 1.262 1.185 4277925 1262 4.T.-;84.AT.- 1.246 0.937 2295261 1236 0.T.-;78.A.T 1.262 0.620 8352817 1263 86.C.- 1.245 0.151 14798078 1237 -29.A.C;76.GG.-A 1.261 0.215 8538048 1264 75.-.G;129.C.A 1.244 0.412 3307911 1238 0.T.-;2.A.G;86.-.G 1.259 0.787 14797557 1265 -29.A.C;75.-.A 1.243 0.320 8132962 1239 75.-.C;87.-.G 1.259 0.464 8538200 1266 75.-.G;133.A.0 1.242 0.440 1-0 n 10181383 1240 18.-.G;75.CG.-A 1.258 0.523 4283490 1267 4.T.-;82.-.0 1.242 0.687 1-3 8197001 1241 86.-.A 1.257 0.487 1865218 1268 0.TT.--;73.A.- 1.241 0.704 cp n.) o 10309927 1242 17.-.T;76.G.-;78.A.T 1.257 0.745 6525015 1269 17.-.G;75.-.A 1.241 0.979 n.) o 2301271 1243 0.T.-;73.AT.-C 1.256 0.811 10181717 1270 18.-.G;76.GG.-A 1.240 1.138 c,.) o 13853791 1244 -14.A.C;75.-.G 1.255 0.426 6458686 1271 16.-.C;76.GG.-C 1.240 0.874 un =
un index SEQ ID muts lindexed index SEQ
ID muts lindexed MI 95% CI
MI 95% CI
NO NO

9978404 1272 19.-.G;86.-.A 1.239 0.802 10561000 1298 15.-.T;76.G.-;78.A.T 1.219 0.648 n.) o 9631659 1273 16. 3318946 1299 0.T.-;2.A.G;81.GA.-T 1.218 0.705 n.) o 1.238 1.158 .CTCATTACTTTG
10565555 1300 15.-.T;75.CG.-T 1.218 1.207 .6.

1938525 1274 0.TT.--;2.A.C;77.GA.--1.235 0.873 oe 2644619 1301 2.A.C;0.T.-;72.-.0 1.218 0.643 2 1907202 1275 0.TTA.---;3.C.A;87.-.G 1.235 0.900 12112275 1302 2.A.-;74.T.G 1.217 0.653 2315524 1276 0.T.-;55.-.T 1.234 0.655 1862409 1303 0.TT.--;76.-.G 1.217 0.889 8531688 1277 75.-.G;89.-.A 1.234 0.685 7189944 1304 27.-.A;78.-.T 1.216 1.075 14798356 1278 -29.A.C;76.-.A 1.233 0.885 6126842 1305 14.-.A;78.-.0 1.216 0.768 8590491 1279 73.A.G 1.233 0.307 8543659 1306 75.-.G;88.-.G 1.215 0.655 3335980 1280 2.A.G;0.T.-;75.C.- 1.231 0.616 2684568 1307 2.A.C;0.T.- 1.213 0.265 2695420 1281 0.T.-;2.A.C;91.AA.-G 1.231 1.033 2697264 1308 2.A.C;0.T.-;89.A.G 1.213 1.022 P
3307298 1282 0.T.-;2.A.G;87.-.T
1.231 0.519 4285424 1309 4.T.-;82.A.G 1.211 1.094 , 2560220 1283 0.T.-;2.A.C;14.-.A
1.231 0.622 .
N)n.) 4298510 1310 4.T.-;78.A.-;80.A.- 1.209 0.668 15165185 1284 -29.A.G;87.-.G 1.231 0.270 3594929 1311 2.-.A;87.-.T 1.209 0.739 "
.
12718005 1285 0.-.T;74.-.G
1.231 0.871 r., , 10310746 1312 17.-.T;76.-.T 1.209 0.919 ' , 10058332 1286 19.-.T;55.-.G
1.230 1.084 r., , 6535421 1313 17.-.G;74.-.T 1.208 0.927 .
8532180 1287 75.-.G;98.-.A 1.229 0.749 2738172 1314 0.T.-;2.A.C;73.-.G 1.208 1.035 7242912 1288 27.-.C;90.-.G 1.229 0.949 1942201 1315 0.TT.--;2.A.C;87.-.G 1.208 0.973 8105731 1289 76.GG.-A;131.A.0 1.228 0.230 8518877 1316 76.GG.-T;121.C.A 1.207 0.182 2748293 1290 2.A.C;0.T.-;66.C.- 1.228 0.985 15159780 1317 -29.A.G;75.-.A 1.206 0.316 3026215 1291 1.TA.--;77.GA.--;83.A.T 1.227 0.998 2290805 1318 0.T.-;79.GAGAAA.TTTCTC 1.204 0.869 1938157 1292 0.TT.--;2.A.C;77.-.A 1.226 0.831 2399086 1319 1.-.A;76.GG.-A 1.204 0.484 1-0 11775381 1293 2.-.C;76.G.-1.225 0.596 n 1974829 1320 0.T.C;76.GG.-A 1.204 0.421 1-3 15161003 1294 -29.A.G;76.G.- 1.224 0.295 1192019 1321 -15.T.G;0.T.-;2.A.0 1.204 0.303 cp n.) 14811016 1295 -29.A.C;78.-.0 1.223 0.273 8565342 1322 75.CG.-T;132.G.0 1.202 0.287 2 o 7237431 1296 27.-.C;72.-.A
1.222 1.143 -a 5 8357813 1323 87.-.G;132.G.0 1.202 0.284 c,.) 4220887 1297 4.T.-;72.-.0 1.220 0.666 o un 14647197 1324 -29.A.C;0.T.-;2.A.C;75.-.G 1.200 0.596 =
un index SEQ ID muts lindexed index SEQ
ID muts lindexed MI 95% CI
MI 95% CI
NO NO

10192426 1325 18.-.G;86.C.- 1.198 0.846 15241255 1352 -29.A.G;2.A.-;75.-.G 1.186 0.444 n.) o 2239077 1326 0.T.-;65.GC.-A 1.197 0.828 6362433 1353 17.-.A;76.GG.-A 1.186 0.851 n.) o iz..1 12185807 1327 2.A.-;80.A.-;82.A.- 1.196 1.148 2059902 1354 0.TT.--;2.A.G;74.-.T 1.186 1.169 .6.
--.1 oe 14921338 1328 -29.A.C;2.A.-;76.GG.-T 1.195 0.591 14799744 1355 -29.A.C;77.-.A 1.186 0.192 wc'e 1909484 1329 0.TTA.---;3.C.A;74.-.T 1.195 0.900 8118273 1356 76.GG.-C;132.G.T 1.185 0.630 10067367 1330 19.-.T;74.-.G 1.194 0.704 4278865 1357 4.T.-;84.-.T 1.184 1.108 8406855 1331 82.A.-;84.A.T 1.194 0.570 10065094 1358 19.-.T;72.-.0 1.183 0.675 3084704 1332 1.TA.--;15.-.T 1.194 0.639 8561350 1359 74.-.T;87.-.G 1.182 0.393 8117630 1333 76.GG.-C;121.C.A 1.194 0.494 15160423 1360 -29.A.G;76.GG.-C 1.181 0.556 14813162 1334 -29.A.C;76.-.T 1.194 0.312 2994738 1361 1.TA.--;74.T.G 1.181 0.980 10086912 1335 19.-.T;78.A.- 1.194 0.527 15058565 1362 -29.A.G;0.T.-;2.A.0 1.180 0.270 P
8565389 1336 75.CG.-T;132.G.T 1.193 0.299 12222182 1363 2.A.-;65.GC.-T 1.180 0.796 ,..
, r., n.) 6627225 1337 18.C.-;76.GG.-T
1.192 0.551 2881480 1364 1.-.C;74.T.- 1.180 0.538 ' .3 ,..
.6.
8485326 1338 76.-.G;86.-.0 1.192 0.494 10193035 1365 18.-.G;86.-.G 1.178 0.685 "
r., , 1853928 1339 0.TT.--;79.G.- 1.192 0.949 6459089 1366 16.-.C;75.-.0 1.178 0.589 ' , r., , 12437875 1340 1.TAC.---;76.-.G 1.192 0.823 10298749 1367 17.-.T;89.-.0 1.178 0.684 10182569 1341 18.-.G;75.-.0 1.192 0.877 8490381 1368 76.-.G;132.G.0 1.177 0.336 6584325 1342 18.-.A;76.-.G 1.191 0.956 12306660 1369 2.A.-;18.-.G 1.177 0.435 8638758 1343 66.CT.-G;76.-.G 1.190 0.454 8124036 1370 75.-.C;98.-.A 1.177 0.499 6460324 1344 16.-.C;79.G.- 1.190 0.494 2893687 1371 1.-.C;88.-.T 1.175 0.780 8365015 1345 87.C.T 1.190 0.873 6305247 1372 16.-.A;77.GA.-- 1.174 0.634 8490408 1346 76.-.G 1.190 0.320 7248579 1373 27.-.C;83.-.T 1.174 1.084 1-0 n 6525955 1347 17.-.G;75.-.0 1.188 1.100 2883890 1374 1.-.C;75.-.0 1.173 0.614 1-3 6460105 1348 16.-.C;76.G.-;78.A.0 1.188 0.685 10183041 1375 18.-.G;76.G.- 1.173 0.967 cp n.) o 6112043 1349 14.-.A;75.-.0 1.188 0.773 2696443 1376 0.T.-;2.A.C;89.A.0 1.173 0.977 n.) o 1978266 1350 0.T.C;86.C.- 1.186 0.483 15239681 1377 -29.A.G;2.A.-;76.G.- 1.173 0.487 c,.) o 8636881 1351 66.CT.-G;87.-.G 1.186 0.214 8087771 1378 74.-.G;87.-.G 1.173 0.426 un =
un index SEQ ID muts lindexed index SEQ
ID muts lindexed MI 95% CI
MI 95% CI
NO NO

10285497 1379 17.-.T;79.G.- 1.172 0.930 8093224 1406 75.-.A;129.C.A 1.151 0.273 n.) o 8118258 1380 76.GG.-C;133.A.0 1.171 0.499 3323632 1407 2.A.G;0.T.-;78.AG.-C 1.151 0.849 n.) o iz..1 8141939 1381 76.G.-;121.C.A 1.171 0.257 14663326 1408 -29.A.C;0.T.-;2.A.G;75.-.G 1.150 0.600 .6.
--.1 oe 8066677 1382 74.T.- 1.169 0.240 1936729 1409 0.TT.--;2.A.C;74.-.G 1.150 1.030 wc'e 8558553 1383 74.-.T;132.G.T 1.168 0.294 1977130 1410 0.T.0 1.148 0.707 6469022 1384 16.-.C;89.-.0 1.168 0.468 8141742 1411 120.C.A;76.G.- 1.148 0.267 1046356 1385 -17.C.A;75.-.G 1.167 0.335 1908681 1412 0.TTA.---;3.C.A;76.-.G 1.148 0.965 10532753 1386 15.-.T;89.-.A 1.166 0.942 3017898 1413 1.TA.--;89.A.G 1.148 0.737 2706855 1387 2.A.C;0.T.-;83.-.G 1.166 0.619 3340495 1414 0.T.-;2.A.G;73.A.0 1.148 1.096 12194678 1388 2.A.-;78.A.G 1.165 0.915 2254255 1415 0.T.-;75.CG.-A 1.147 0.701 12126149 1389 2.A.-;77.-.0 1.164 0.392 11953402 1416 2.AC.--;4.T.C;76.GG.-C 1.145 1.093 P
3039439 1390 1.TA.--;70.-.T 1.163 1.008 2684619 1417 0.T.-;2.A.C;132.G.T 1.145 0.260 ,..
, r., n.) 8123371 1391 75.-.C;87.-.A
1.162 0.505 10314306 1418 17.-.T;73.AT.-C 1.144 1.029 ' .3 ,..
un 15160286 1392 -29.A.G;76.-.A 1.162 0.722 10559572 1419 15.-.T;78.A.G 1.144 0.579 "
r., , 8758541 1393 55.-.T;80.A.- 1.161 0.587 2630318 1420 2.A.C;0.T.-;66.CT.-A 1.144 0.534 ' , r., , 12433294 1394 1.TAC.---;79.G.- 1.161 0.560 1943847 1421 0.TT.--;2.A.C;81.GA.-T 1.143 0.765 14801714 1395 -29.A.C;87.-.A 1.160 0.841 4270685 1422 4.T.-;90.-.T 1.142 1.061 15058156 1396 2.A.C;0.T.-;-29.A.G;76.G.- 1.159 0.397 8066737 1423 74.T.-;131.A.0 1.142 0.298 2298993 1397 0.T.-;75.C.- 1.158 0.419 6101577 1424 14.-.A;55.-.G 1.142 0.632 13100965 1398 -1.GT.--;78.A.- 1.158 0.371 4279604 1425 4.T.-;82.A.- 1.141 0.866 8438445 1399 77.GA.--;83.A.T 1.156 0.839 2284176 1426 0.T.-;83.-.G 1.141 0.574 8519469 1400 76.GG.-T;132.G.0 1.156 0.148 6480468 1427 16.-.C;70.-.T 1.140 0.614 1-0 n 8569101 1401 75.CGG.-TT 1.155 0.217 2640116 1428 0.T.-;2.A.C;71.-.0 1.137 0.936 1-3 4310993 1402 4.T.-;73.AT.-C 1.153 0.454 10194587 1429 18.-.G;82.AA.-C 1.137 0.867 cp n.) o 9971050 1403 19.-.G;72.-.0 1.153 0.725 15456465 1430 -30.C.G;75.-.G 1.136 0.421 n.) o 2996647 1404 1.TA.--;75.CG.-A 1.152 0.812 3432602 1431 0.T.-;2.A.G;18.-.G 1.136 0.359 c,.) o 8561305 1405 74.-.T;86.C.- 1.151 0.238 8345813 1432 89.-.T 1.135 0.634 un =
un index SEQ ID muts lindexed index SEQ
ID muts lindexed MI 95% CI
MI 95% CI
NO NO

3023247 1433 1.TA.--;83.-.T 1.135 0.960 3027775 1460 1.TA.--;80.AG.-T 1.121 0.673 n.) o 10472698 1434 16.C.-;76.-.G 1.134 0.911 10549691 1461 15.-.T;82.A.- 1.120 0.844 n.) o iz..1 1855129 1435 0.TT.--;88.G.- 1.133 0.759 8558571 1462 74.-.T;131.A.0 1.119 0.242 .6.
--.1 oe 9993029 1436 19.-.G;78.A.- 1.133 0.793 12210725 1463 2.A.-;73.AT.-G 1.119 0.805 wc'e 15168776 1437 -29.A.G;76.GG.-T 1.132 0.227 6462677 1464 16.-.C;86.-.0 1.118 0.994 2464359 1438 1.TA.--;3.C.A;82.A.-;84.A.G 1.132 1.057 2281811 1465 0.T.-;86.CC.-T 1.118 0.883 12156161 1439 2.A.-;98.-.T 1.131 0.852 8496336 1466 78.A.-;80.A.- 1.117 0.515 8544614 1440 75.-.G;82.A.- 1.131 0.458 3038148 1467 1.TA.--;73.A.0 1.117 0.862 2278784 1441 0.T.-;89.A.G 1.130 0.932 10199335 1468 75.-.G;127.T.G 1.116 0.444 4229697 1442 4.T.-;75.CG.-A 1.129 1.031 14801930 1469 -29.A.C;88.G.- 1.115 0.262 6461360 1443 16.-.C;82.-.A 1.129 0.609 2885740 1470 1.-.C;81.GA.-C 1.115 0.689 P
8128601 1444 133.A.C;75.-.0 1.129 0.316 8436871 1471 81.GA.-T 1.115 0.274 ,..
, r., n.) A 6362009 1445 17.-.;74.-.G
1.128 0.792 6533591 1472 17.-.G;78.-.0 1.115 0.880 ' .3 ,..
o 14806733 1446 -29.A.C;86.C.- 1.128 0.128 8508461 1473 78.A.T 1.115 0.523 "
r., , 1937160 1447 0.TT.--;2.A.C;76.GG.-A 1.126 1.000 2303258 1474 0.T.-;70.-.T 1.114 0.865 ' , r., , 4311644 1448 4.T.-;73.A.0 1.126 0.593 10200479 1475 18.-.G;75.CG.-T 1.113 0.732 1863149 1449 0.TT.--;76.GG.-T 1.126 0.643 8142460 1476 76.G.-;126.C.A 1.111 0.288 15169751 1450 -29.A.G;74.-.T 1.126 0.265 8490449 1477 76.-.G;132.G.T 1.111 0.315 14811726 1451 -29.A.C;76.-.G 1.126 0.338 1862090 1478 0.TT.--;78.A.- 1.111 0.800 6480066 1452 16.-.C;73.AT.-G 1.125 0.918 8105143 1479 76.GG.-A;121.C.A 1.111 0.256 3014440 1453 1.TA.--;98.-.T 1.125 0.945 10204124 1480 18.-.G;65.GC.-T 1.110 0.661 6473404 1454 16.-.C;82.AA.-T 1.125 0.450 2696979 1481 0.T.-;2.A.C;88.-.G 1.110 0.607 1-0 n 7179375 1455 27.-.A;73.-.A 1.123 1.119 1246393 1482 -15.T.G;76.GG.-A 1.110 0.194 1-3 12303885 1456 2.A.-;19.-.T 1.123 0.456 4277641 1483 4.T.-;84.-.0 1.109 1.085 cp n.) o 2267762 1457 0.T.-;98.-.A 1.122 0.679 12163684 1484 2.A.-;88.-.G 1.109 0.570 n.) o 10318319 1458 17.-.T;66.CT.-G 1.122 1.050 3643882 1485 3.CT.-A;76.GG.-A 1.109 0.785 c,.) o 8093357 1459 75.-.A;132.G.T 1.121 0.315 6461122 1486 16.-.C;81.GA.-C 1.108 0.626 un =
un index SEQ ID muts lindexed index SEQ
ID muts lindexed MI 95% CI
MI 95% CI
NO NO

14645694 1487 2.A.C;0.T.-;-29.A.0 1.108 0.268 10194914 1514 18.-.G;82.AA.-G 1.095 0.926 n.) o 2678659 1488 0.T.-;2.A.C;98.-.A 1.108 0.376 1041972 1515 -17.C.A;76.G.- 1.094 0.260 n.) o iz..1 2295085 1489 0.T.-;77.GA.--;80.A.T 1.108 0.695 8537811 1516 75.-.G;126.C.A 1.094 0.416 .6.
--.1 oe 8127785 1490 75.-.C;120.C.A 1.107 0.299 3020817 1517 1.TA.--;84.AT.-- 1.094 1.006 wc'e 8357871 1491 87.-.G;132.G.T 1.107 0.336 2887379 1518 1.-.C;86.-.0 1.093 0.650 12090020 1492 2.A.-;66.CT.-A 1.106 0.760 1854285 1519 0.TT.--;77.GA.-- 1.093 0.836 3079463 1493 1.TA.--;19.-.T 1.105 0.424 8357326 1520 87.-.G;121.C.A 1.093 0.228 10277558 1494 17.-.T;72.-.G 1.105 0.335 8128534 1521 75.-.C;130.T.G 1.092 0.292 2694724 1495 0.T.-;2.A.C;92.A.T 1.102 0.929 1947291 1522 0.TT.--;2.A.C;73.A.- 1.092 1.083 3135565 1496 1.T.G;3.C.-;75.C.- 1.102 0.673 12432721 1523 1.TAC.---;76.GG.-C 1.091 0.425 6304328 1497 16.-.A;75.-.0 1.102 0.655 1252779 1524 -15.T.G;75.-.G 1.091 0.436 P
2708067 1498 2.A.C;0.T.-;83.-.T 1.102 0.859 3588353 1525 2.-.A;86.-.0 1.090 0.473 ,..
, r., n.) A 6469331 1499 16.-.C;89..-1.101 0.791 2900664 1526 1.-.C;76.GG.-T 1.090 0.928 ' .3 ,..
--.1 10073526 1500 19.-.T;90.T.- 1.101 0.917 8076983 1527 74.T.G 1.090 0.516 "
r., , 3017595 1501 1.TA.--;89.AT.-G 1.101 0.904 2300899 1528 0.T.-;73.-.0 1.088 0.922 ' , r., , 3031194 1502 1.TA.--;78.A.G 1.100 1.042 12202788 1529 2.A.-;75.-.G;132.G.0 1.087 0.397 12123777 1503 2.A.-;76.G.-;132.G.0 1.100 0.426 10070325 1530 19.-.T;77.-.A 1.085 0.602 15451300 1504 -30.C.G;76.G.- 1.100 0.258 14685826 1531 -29.A.C;4.T.-;76.G.- 1.085 0.875 8105041 1505 76.GG.-A;120.C.A 1.100 0.198 14351033 1532 -25.A.C;75.-.G 1.085 0.402 2894267 1506 1.-.C;87.-.T 1.099 0.722 8607376 1533 73.A.T 1.084 0.466 2998547 1507 1.TA.--;76.GG.-C 1.099 0.772 12439360 1534 1.TAC.---;73.A.- 1.084 0.785 3022051 1508 1.TA.--;83.-.0 1.099 0.800 12718596 1535 0.-.T;75.-.A 1.083 0.730 1-0 n 8512487 1509 76.G.-;78.A.T 1.098 0.434 2712801 1536 2.A.C;0.T.-;82.A.T 1.083 1.030 1-3 2285757 1510 0.T.-;82.AA.-C 1.098 0.581 6613293 1537 18.C.-;77.-.0 1.082 0.704 cp n.) o 6531470 1511 17.-.G;87.-.G 1.097 0.892 8480766 1538 78.A.- 1.081 0.244 n.) o 3461447 1512 0.TTAC.----;78.A.- 1.097 1.032 2414074 1539 1.-.A;75.CG.-T 1.078 0.690 c,.) o 6475031 1513 16.-.C;78.-.0 1.096 0.623 8105662 1540 76.GG.-A;132.G.0 1.078 0.266 un =
un index SEQ ID muts lindexed index SEQ
ID muts lindexed MI 95% CI
MI 95% CI
NO NO

2282078 1541 0.T.-;84.AT.-- 1.078 1.018 2684598 1568 0.T.-;2.A.C;133.A.0 1.064 0.264 n.) o 8096091 1542 75.-.A;86.C.- 1.078 0.285 1806606 1569 -3.TAGT.----;76.G.- 1.063 0.955 n.) o 442111 1543 -27.C.A;76.GG.-C 1.078 0.495 6112609 1570 14.-.A;76.G.- 1.063 0.690 .6.

oe 12161656 1544 2.A.-;91.A.G 1.076 0.678 8128619 1571 75.-.C;132.G.T 1.063 0.341 wc'e 9997135 1545 19.-.G;75.CG.-T 1.076 0.618 2263869 1572 0.T.-;85.-.G 1.062 1.017 6480747 1546 16.-.C;73.A.- 1.074 0.613 8519538 1573 76.GG.-T;131.A.0 1.061 0.210 8066659 1547 74.T.-;132.G.0 1.074 0.263 15167837 1574 -29.A.G;78.A.- 1.061 0.247 4265165 1548 4.T.-;99.-.G 1.073 0.742 8539891 1575 113.A.C;75.-.G 1.061 0.380 8212888 1549 86.-.C;132.G.T 1.072 0.490 6110621 1576 14.-.A;75.-.A 1.060 0.621 10532402 1550 15.-.T;88.GA.-C 1.071 0.565 4012102 1577 3.-.C;76.GG.-A 1.059 1.032 2897244 1551 1.-.C;81.GA.-T 1.071 0.381 14644765 1578 -29.A.C;0.T.-;2.A.C;76.GG.-A
1.059 0.330 P
2274809 1552 0.T.-;98.-.T 1.071 0.702 6114928 1579 14.-.A;87.-.A 1.058 0.886 , r., n.) A 3584484 1553 2.-.;76.GG.-C
1.071 0.859 1858781 1580 0.TT.--;87.-.T 1.058 0.825 ' .3 oe 12115802 1554 2.A.-;75.CG.-A 1.070 0.736 10090936 1581 19.-.T;75.CG.-T 1.056 0.659 "
r., , 3349186 1555 2.A.G;0.T.-;66.CT.-G 1.070 0.943 2002673 1582 0.TTA.---;86.-.0 1.055 0.913 ' , r., , 3314448 1556 0.T.-;2.A.G;82.A.-;84.A.T 1.069 0.670 1937274 1583 0.TT.--;2.A.C;76.-.A 1.055 0.766 2882882 1557 1.-.C;76.GG.-A 1.069 0.641 1946930 1584 2.A.C;0.TT.--;73.AT.-G 1.054 1.042 8112365 1558 132.G.C;76.-.A 1.068 0.642 8564806 1585 75.CG.-T;121.C.A 1.054 0.274 8118289 1559 76.GG.-C;131.A.0 1.068 0.672 14646874 1586 -29.A.C;0.T.-;2.A.C;78.A.- 1.053 0.595 2684538 1560 0.T.-;2.A.C;132.G.0 1.068 0.292 3279449 1587 2.A.G;0.T.-;86.-.A 1.053 0.589 3305808 1561 2.A.G;0.T.-;86.C.- 1.067 0.815 10183929 1588 18.-.G;79.G.- 1.052 0.658 12141962 1562 2.A.-;98.-.A 1.067 0.769 4281239 1589 4.T.-;83.-.G 1.052 0.864 1-0 n 8629287 1563 66.CT.-G;87.-.A 1.067 0.521 8636987 1590 66.CT.-G;87.-.T 1.052 0.463 1-3 10548927 1564 15.-.T;84.-.G 1.066 0.949 2684414 1591 129.C.A;2.A.C;0.T.- 1.051 0.312 cp n.) o 12437589 1565 1.TAC.---;78.-.0 1.066 1.010 10567800 1592 15.-.T;70.-.T 1.050 0.621 n.) o -a 5 8494451 1566 76.-.G;87.-.G 1.065 0.356 12183487 1593 2.A.-;77.GA.--;83.A.T 1.049 0.987 c,.) o 8148054 1567 76.G.-;87.-.G 1.065 0.414 3429655 1594 0.T.-;2.A.G;19.-.T 1.049 0.495 un =
un index SEQ ID muts lindexed index SEQ
ID muts lindexed MI 95% CI
MI 95% CI
NO NO

15168064 1595 -29.A.G;76.-.G 1.048 0.302 15059527 1622 -29.A.G;0.T.-;2.A.C;75.-.G 1.033 0.531 n.) o 8579268 1596 73.A.0 1.048 0.683 8127925 1623 75.-.C;121.C.A 1.032 0.246 n.) o 12725378 1597 0.-.T;86.-.A 1.047 0.366 8069875 1624 74.T.-;87.-.G 1.032 0.583 .6.

oe 12133179 1598 2.A.-;85.TC.-- 1.047 0.820 4210905 1625 4.T.-;66.CT.-A 1.032 0.842 wc'e 12169171 1599 2.A.-;87.C.T 1.047 0.600 393375 1626 -27.C.A;0.T.-;2.A.0 1.031 0.249 1974530 1600 0.T.C;74.-.G 1.045 0.682 6469193 1627 16.-.C;88.-.G 1.030 0.736 3276852 1601 2.A.G;0.T.-;81.GA.-C 1.045 0.975 12723788 1628 0.-.T;77.GA.-- 1.030 0.436 2277126 1602 0.T.-;91.A.-;93.A.G 1.044 0.955 1975104 1629 0.T.C;75.-.0 1.030 0.579 2668148 1603 0.T.-;2.A.C;80.-.A 1.043 0.586 447486 1630 -27.C.A;74.-.T 1.030 0.222 1946365 1604 0.TT.--;2.A.C;74.-.T 1.043 1.041 2304326 1631 0.T.-;73.A.T 1.029 0.531 10086224 1605 19.-.T;78.AG.-C 1.043 0.736 8480805 1632 78.A.-;132.G.T 1.029 0.245 P
6474902 1606 16.-.C;78.AG.-C 1.042 0.503 10289207 1633 17.-.T;89.-.A 1.026 0.760 ,..
, r., n.) A 3001790 1607 1.T.--;77.-.0 1.042 0.684 10541758 1634 15.-.T;99.-.G 1.026 0.736 ' .3 ,..
o 6463023 1608 16.-.C;89.-.A 1.042 0.830 8580639 1635 73.-TC.G-- 1.026 0.359 "
r., , 8470293 1609 78.-.C;132.G.T 1.042 0.300 2129400 1636 0.TTA.---;3.C.G;74.-.T 1.026 1.011 ' , r., , 3134206 1610 1.T.G;3.C.- 1.041 0.793 8142671 1637 76.G.-;128.T.G 1.026 0.290 10203551 1611 18.-.G;66.CT.-G 1.040 0.787 12726231 1638 0.-.T;88.G.- 1.026 0.405 8629503 1612 66.CT.-G;86.-.0 1.039 0.370 10288957 1639 17.-.T;88.GA.-C 1.025 0.602 13846013 1613 -14.A.C;76.G.- 1.038 0.247 2982939 1640 1.TA.--;65.GC.-A 1.025 0.854 2263715 1614 0.T.-;85.TC.-G 1.038 0.802 8357852 1641 87.-.G;133.A.0 1.024 0.267 10560681 1615 15.-.T;78.A.T 1.038 0.677 6626305 1642 18.C.-;76.-.G 1.024 0.941 1253221 1616 -15.T.G;75.CG.-T 1.038 0.213 15167605 1643 -29.A.G;78.-.0 1.024 0.228 1-0 n 10556907 1617 15.-.T;78.AG.-C 1.037 1.020 3273923 1644 2.A.G;0.T.-;79.G.- 1.022 0.761 1-3 3319204 1618 0.T.-;2.A.G;77.GA.--;83.A.T 1.036 0.978 10553626 1645 15.-.T;82.AA.-T 1.020 0.844 cp n.) o 2277677 1619 0.T.-;91.AA.-G 1.035 0.945 3029129 1646 1.TA.--;78.A.0 1.018 0.493 n.) o -a 5 3044097 1620 1.TA.--;65.GC.-T 1.034 0.777 3133667 1647 1.T.G;3.C.-;76.G.- 1.018 0.664 c,.) o 2728986 1621 0.T.-;2.A.C;76.GG.--;78.A.T 1.033 0.961 14921066 1648 -29.A.C;2.A.-;78.A.- 1.018 0.654 un =
un index SEQ ID muts lindexed index SEQ
ID muts lindexed MI 95% CI
MI 95% CI
NO NO

14806598 1649 -29.A.C;88.-.T 1.017 0.327 12174360 1676 2.A.-;83.-.0 1.002 0.612 n.) o 8139512 1650 115.T.G;76.G.- 1.017 0.260 442458 1677 -27.C.A;76.G.- 1.001 0.255 n.) o iz..1 8636794 1651 66.CT.-G;86.C.- 1.017 0.224 15162537 1678 -29.A.G;86.-.0 1.000 0.512 .6.
--.1 oe 8127584 1652 75.-.C;119.C.A 1.017 0.258 2991036 1679 1.TA.--;72.-.0 0.999 0.524 wc'e 4311933 1653 4.T.-;73.-.G 1.016 0.722 8489557 1680 76.-.G;120.C.A 0.999 0.235 6471359 1654 16.-.C;83.-.0 1.016 0.690 2704195 1681 0.T.-;2.A.C;84.A.G 0.999 0.779 12433542 1655 1.TAC.---;77.GA.-- 1.015 0.963 12746931 1682 0.-.T;78.AG.-T 0.999 0.695 8093303 1656 75.-.A;132.G.0 1.014 0.287 8544289 1683 75.-.G;86.-.G 0.998 0.330 1246761 1657 -15.T.G;75.-.0 1.014 0.245 8490052 1684 76.-.G;126.C.A 0.998 0.284 1943763 1658 0.TT.--;2.A.C;82.AA.-T 1.013 0.876 3003857 1685 1.TA.--;81.GA.-C 0.997 0.622 4158980 1659 4.T.-;16.-.0 1.012 0.731 2683589 1686 0.T.-;2.A.C;121.C.A 0.997 0.259 P
8470306 1660 78.-.C;131.A.0 1.012 0.269 8565256 1687 75.CG.-T;129.C.A 0.996 0.264 ,..
, r., n.) 8069089 1661 74.T.-;98.-.T
1.012 0.754 2684649 1688 0.T.-;2.A.C;131.A.0 0.995 0.272 ' .3 .6.
,..
o 12438882 1662 1.TAC.---;75.CG.-T 1.012 0.646 10192242 1689 18.-.G;88.-.T 0.995 0.989 "
r., , 8338521 1663 89.AT.-G 1.010 0.922 8128468 1690 75.-.C;129.C.A 0.995 0.262 ' , r., , 10088951 1664 19.-.T;76.-.T 1.010 0.995 3255338 1691 2.A.G;0.T.-;72.-.0 0.994 0.842 12163085 1665 2.A.-;89.A.0 1.010 1.006 7829410 1692 55.-.G;75.-.0 0.994 0.860 8479927 1666 78.A.-;121.C.A 1.008 0.198 15162331 1693 -29.A.G;87.-.A 0.993 0.691 10196772 1667 18.-.G;78.A.0 1.007 0.606 8212834 1694 86.-.C;132.G.0 0.992 0.467 8552295 1668 75.C.-;87.-.G 1.006 0.446 13222300 1695 2.A.G;-3.TAGT.----;76.G.- 0.991 0.723 4027916 1669 3.-.C;74.-.T 1.006 0.888 8470255 1696 78.-.C;132.G.0 0.991 0.219 8489338 1670 76.-.G;119.C.A 1.005 0.338 2661937 1697 132.G.C;2.A.C;0.T.-;76.G.- 0.990 0.390 n 446968 1671 -27.C.A;76.GG.-T 1.005 0.187 2670761 1698 0.T.-;2.A.C;85.TCC.--- 0.990 0.720 1-2049927 1672 0.TT.--;2.A.G;88.G.- 1.005 0.953 11776916 1699 2.-.C;87.-.A 0.989 0.938 cp n.) o 8598621 1673 70.-.T;87.-.G 1.004 0.383 12747759 1700 0.-.T;77.-.T 0.989 0.938 n.) o 8600573 1674 73.A.-;86.-.0 1.004 0.369 15165085 1701 -29.A.G;86.C.- 0.987 0.176 c,.) o 8473900 1675 78.A.0 1.003 0.272 8212745 1702 86.-.C;129.C.A 0.987 0.509 un =
un index SEQ ID muts lindexed index SEQ
ID muts lindexed MI 95% CI

NO NO

2989789 1703 1.TA.--;72.-.A 0.986 0.659 14646928 1730 -29.A.C;0.T.-;2.A.C;76.-.G 0.975 0.273 n.) o 6531564 1704 17.-.G;87.-.T 0.985 0.962 8212907 1731 86.-.C;131.A.0 0.975 0.470 n.) o iz..1 12436169 1705 1.TAC.---;87.-.G 0.984 0.678 13097486 1732 -1.GT.--;75.-.0 0.974 0.347 .6.
--.1 oe 3311127 1706 2.A.G;0.T.-;82.A.- 0.984 0.759 3272148 1733 2.A.G;0.T.-;77.-.A 0.974 0.592 wc'e 2264270 1707 0.T.-;86.CC.-A 0.983 0.775 8557995 1734 74.-.T;121.C.A 0.973 0.210 10091719 1708 19.-.T;73.AT.-G 0.982 0.402 8142576 1735 76.G.-;127.T.G 0.973 0.375 8143233 1709 76.G.-;123.A.0 0.982 0.226 14816291 1736 -29.A.C;73.A.- 0.972 0.232 1248077 1710 -15.T.G;86.-.0 0.981 0.619 10080185 1737 19.-.T;89.-.0 0.971 0.565 12716866 1711 0.-.T;74.T.- 0.981 0.501 1904247 1738 0.TTA.---;3.C.A;75.-.A 0.970 0.749 3303133 1712 2.A.G;0.T.-;89.-.0 0.980 0.929 6460821 1739 16.-.C;77.GA.-- 0.970 0.637 9974910 1713 19.-.G;76.GG.-C 0.980 0.702 12738126 1740 0.-.T;87.-.T 0.968 0.578 P
8143415 1714 76.G.-;122.A.0 0.980 0.247 8357730 1741 87.-.G;129.C.A 0.968 0.270 ,..
, r., n.) 1981670 1715 0.T.C;74.-.T
0.980 0.590 12187919 1742 2.A.-;79.GA.-T 0.968 0.963 ' .3 .6.
,..
1--, 2302384 1716 0.T.-;73.AT.-G 0.978 0.565 14644862 1743 -29.A.C;0.T.-;2.A.C;76.GG.-C 0.967 0.512 r., , 1809039 1717 -3.TAGT.----;78.A.- 0.978 0.801 13101334 1744 -1.GT.--;76.GG.-T 0.967 0.377 ' , r., , 13139359 1718 -1.G.-;2.A.0 0.978 0.275 12437308 1745 1.TAC.---;80.A.- 0.966 0.933 8538659 1719 75.-.G;122.A.0 0.978 0.392 2672055 1746 0.T.-;2.A.C;86.C.A 0.966 0.590 2651461 1720 0.T.-;2.A.C;74.T.G 0.977 0.582 6304109 1747 16.-.A;76.GG.-C 0.966 0.672 3028256 1721 1.TA.--;79.GA.-T 0.977 0.767 12214091 1748 2.A.-;73.A.T 0.966 0.602 444970 1722 -27.C.A;87.-.G 0.976 0.225 8511126 1749 76.G.-;78.AG.TC 0.965 0.454 2271218 1723 132.G.T;0.T.- 0.976 0.376 10473646 1750 16.C.-;76.GG.-T 0.965 0.499 13101059 1724 -1.GT.--;76.-.G 0.976 0.320 8561622 1751 74.-.T;82.A.- 0.965 0.362 1-0 n 15169928 1725 -29.A.G;75.CG.-T 0.976 0.276 1981516 1752 0.T.C;75.C.- 0.964 0.525 1-3 6454149 1726 16.-.C;72.-.0 0.976 0.472 4300894 1753 4.T.-;77.G.T 0.964 0.236 cp n.) o 8519506 1727 76.GG.-T;133.A.0 0.976 0.183 8084158 1754 74.-.G 0.964 0.402 n.) o 1936400 1728 0.TT.--;2.A.C;74.T.- 0.975 0.971 8096194 1755 75.-.A;87.-.T 0.964 0.605 c,.) o 8363289 1729 87.-.T;132.G.T 0.975 0.349 2281085 1756 0.T.-;87.C.T 0.961 0.675 un =
un index SEQ ID muts lindexed index SEQ
ID muts lindexed MI 95% CI
MI 95% CI
NO NO

8063355 1757 74.T.-;86.-.0 0.960 0.507 10523926 1784 15.-.T;76.-.A 0.948 0.739 n.) o 3038327 1758 1.TA.--;73.-.G 0.959 0.854 12742835 1785 0.-.T;81.GA.-T 0.948 0.383 n.) o iz..1 9976817 1759 19.-.G;79.G.- 0.958 0.737 8093342 1786 75.-.A;133.A.0 0.948 0.327 .6.
--.1 oe 13223005 1760 2.A.G;-3.TAGT.---- 0.958 0.837 8490265 1787 76.-.G;129.C.A 0.948 0.322 wc'e 8542589 1761 75.-.G;98.-.T 0.957 0.875 2412848 1788 1.-.A;76.-.T 0.947 0.632 3345006 1762 0.T.-;2.A.G;73.A.T 0.957 0.793 8183422 1789 85.TC.-A 0.947 0.638 4217628 1763 4.T.-;71.-.0 0.956 0.495 2463159 1790 1.TA.--;3.C.A;88.-.T 0.946 0.552 10068711 1764 19.-.T;76.-.A 0.956 0.689 8490433 1791 76.-.G;133.A.0 0.946 0.318 10198139 1765 18.-.G;77.-.T 0.956 0.663 2681222 1792 0.T.-;2.A.C;115.T.G 0.946 0.288 2463484 1766 1.TA.--;3.C.A;87.-.T 0.955 0.695 8480741 1793 78.A.-;132.G.0 0.946 0.202 8490228 1767 76.-.G;128.T.G 0.955 0.305 2663534 1794 0.T.-;2.A.C;77.G.0 0.946 0.861 P
3322121 1768 0.T.-;2.A.G;80.AG.-T 0.955 0.812 8118132 1795 76.GG.-C;129.C.A 0.946 0.373 ,..
, r., n.) 2458850 1769 1.TA.--;3.C.A;79.G.-0.955 0.858 6447398 1796 16.-.C;55.-.G 0.945 0.768 ' .3 .6.
,..
n.) 6626017 1770 18.C.-;78.A.- 0.954 0.611 2285156 1797 0.T.-;82.AA.-- 0.945 0.503 "
r., , 8519520 1771 76.GG.-T;132.G.T 0.954 0.281 8117520 1798 76.GG.-C;120.C.A 0.945 0.413 ' , r., , 1974653 1772 0.T.C;75.-.A 0.954 0.490 8603147 1799 73.A.- 0.945 0.225 2683428 1773 120.C.A;2.A.C;0.T.- 0.954 0.253 8537609 1800 75.-.G;124.T.G
0.944 0.366 4272200 1774 4.T.-;89.A.G 0.954 0.925 2245955 1801 0.T.-;71.-.0 0.944 0.684 8193481 1775 85.TC.-G 0.953 0.701 8161116 1802 79.G.- 0.942 0.264 6557686 1776 18.C.A;75.-.G 0.953 0.330 8536998 1803 75.-.G;119.C.A 0.942 0.370 1860902 1777 0.TT.--;81.GA.-T 0.952 0.515 8537871 1804 75.-.G;127.T.0 0.941 0.334 2717874 1778 2.A.C;0.T.-;80.AG.-T 0.951 0.611 8543767 1805 75.-.G;89.A.- 0.941 0.628 1-0 n 2882024 1779 1.-.C;74.-.G 0.951 0.619 6603080 1806 18.C.-;55.-.G 0.941 0.707 1-3 3273132 1780 0.T.-;2.A.G;77.-.0 0.951 0.397 13850293 1807 -14.A.C;87.-.G 0.940 0.218 cp n.) o 441958 1781 -27.C.A;76.GG.-A 0.949 0.205 1852615 1808 0.TT.--;76.-.A 0.938 0.750 n.) o 14811390 1782 -29.A.C;78.A.- 0.949 0.249 8208020 1809 88.G.-;132.G.0 0.938 0.242 c,.) o 14802094 1783 -29.A.C;86.-.0 0.949 0.461 14918769 1810 -29.A.C;2.A.-;76.GG.-A 0.937 0.353 un =
un index SEQ ID muts lindexed index SEQ
ID muts lindexed MI 95% CI
MI 95% CI
NO NO

8223161 1811 90.-.G 0.937 0.664 14822468 1838 -29.A.C;55.-.T 0.921 0.524 n.) o 2684123 1812 0.T.-;2.A.C;126.C.A 0.936 0.262 8357890 1839 87.-.G;131.A.0 0.921 0.275 n.) o iz..1 2883487 1813 1.-.C;76.GG.-C 0.934 0.884 8485265 1840 76.-.G;88.G.- 0.920 0.453 .6.
--.1 oe 8089075 1814 75.-C.AA 0.934 0.299 14796763 1841 -29.A.C;74.-.0 0.919 0.375 wc'e 13746840 1815 -13.G.T;76.G.- 0.934 0.266 14796493 1842 -29.A.C;74.T.- 0.919 0.249 10179608 1816 18.-.G;73.-.A 0.933 0.587 8558538 1843 74.-.T;133.A.0 0.919 0.281 8357113 1817 87.-.G;119.C.A 0.933 0.238 7247803 1844 27.-.C;86.CC.-G 0.918 0.915 2570963 1818 0.T.-;2.A.C;18.C.- 0.932 0.404 10073442 1845 19.-.T;88.GA.-C 0.918 0.552 6621548 1819 18.C.-;88.-.T 0.932 0.702 12133660 1846 2.A.-;85.TC.-G 0.918 0.916 8543544 1820 75.-.G;89.-.0 0.930 0.331 2572420 1847 0.T.-;2.A.C;19.-.A 0.917 0.558 8158269 1821 79.G.A 0.928 0.860 8555076 1848 74.-.T;88.G.- 0.915 0.377 P
3341556 1822 2.A.G;0.T.-;73.AT.-G 0.928 0.857 10607377 1849 16.C.T;75.-.G 0.915 0.789 ,..
, r., n.) 2683151 1823 119.C.A;2.A.C;O.T.-0.928 0.288 3281290 1850 2.A.G;O.T.-;88.G.-0.915 0.699 ' .3 .6.
,..
8543919 1824 75.-.G;88.-.T 0.926 0.543 12713711 1851 0.-.T;72.-.A 0.915 0.659 "
r., , 2570189 1825 0.T.-;2.A.C;18.-.A 0.926 0.645 15408234 1852 -30.C.G;0.T.-;2.A.0 0.915 0.291 ' , r., , 4015474 1826 3.-.C;86.-.0 0.926 0.838 12722990 1853 0.-.T;79.G.- 0.915 0.499 2731496 1827 0.T.-;2.A.C;75.-.G;132.G.0 0.925 0.518 8105716 1854 76.GG.-A;132.G.T 0.914 0.275 8480834 1828 78.A.-;131.A.0 0.925 0.257 2271180 1855 0.T.- 0.913 0.381 3011827 1829 1.TA.-- 0.923 0.388 10289412 1856 17.-.T;90.-.G 0.913 0.695 8592843 1830 70.-.T;86.-.0 0.923 0.501 14807090 1857 -29.A.C;87.-.T 0.912 0.449 8057655 1831 73.-.A 0.923 0.547 6108421 1858 14.-.A;72.-.0 0.910 0.863 8480787 1832 78.A.-;133.A.0 0.923 0.247 8141461 1859 76.G.-;119.C.A 0.909 0.263 1-0 n 2249456 1833 0.T.-;72.-.G 0.922 0.820 14350324 1860 -25.A.C;76.-.G 0.908 0.330 1-3 8752628 1834 55.-.T;76.GG.-A 0.922 0.503 8538185 1861 130.--T.TAG;133.A.G;75.-.G 0.906 0.421 cp n.) o 2274200 1835 0.T.-;99.-.T 0.921 0.848 8538491 1862 75.-.G;123.A.0 0.906 0.359 n.) o 8142972 1836 76.G.-;131.A.C;133.A.0 0.921 0.258 14292135 1863 -25.A.C;0.T.-;2.A.0 0.905 0.255 c,.) o 1252489 1837 -15.T.G;76.GG.-T 0.921 0.236 2399779 1864 1.-.A;75.-.0 0.904 0.626 un =
un index SEQ ID muts lindexed index SEQ
ID muts lindexed MI 95% CI

NO NO

8142947 1865 76.G.-;131.AG.CC 0.903 0.312 4247573 1892 4.T.-;87.C.A 0.885 0.526 n.) o 8603195 1866 73.A.-;131.A.0 0.902 0.229 6110295 1893 14.-.A;74.-.G 0.884 0.833 n.) o iz..1 3329015 1867 2.A.G;0.T.-;78.-.T 0.901 0.635 6369429 1894 17.-.A;76.-.T 0.884 0.672 .6.
--.1 oe 2457498 1868 1.TA.--;3.C.A;76.-.A 0.901 0.878 6476407 1895 16.-.C;78.-.T 0.883 0.612 wc'e 14799938 1869 -29.A.C;76.G.-;78.A.0 0.901 0.250 2309043 1896 0.T.-;65.GC.-T 0.883 0.649 10194359 1870 18.-.G;82.AA.-- 0.901 0.723 10084280 1897 19.-.T;82.AA.-G 0.883 0.750 2461767 1871 1.TA.--;3.C.A;99.-.G 0.898 0.891 2884850 1898 1.-.C;76.G.-;78.A.0 0.882 0.492 8128631 1872 75.-.C;131.AG.CC 0.898 0.298 2347258 1899 0.T.-;19.-.G 0.880 0.616 6130904 1873 14.-.A;75.CG.-T 0.898 0.809 12737110 1900 0.-.T;88.-.T 0.880 0.357 2885480 1874 1.-.C;77.GA.-- 0.897 0.564 10557558 1901 15.-.T;78.A.0 0.879 0.710 8565409 1875 131.A.C;75.CG.-T 0.896 0.289 1851901 1902 0.TT.--;74.-.G 0.878 0.824 P
8526599 1876 76.-.T;133.A.0 0.895 0.367 6621723 1903 18.C.-;86.C.- 0.877 0.845 ,..
, r., n.) A 8542268 1877 75.-.G;99.-.G
0.895 0.466 10567449 1904 15.-.T;73..G 0.876 0.489 ' .3 .6.
,..
.6.
3296935 1878 0.T.-;2.A.G;98.-.T 0.894 0.819 1863878 1905 0.TT.--;75.C.- 0.876 0.766 "
r., , 8535676 1879 115.T.G;75.-.G 0.892 0.386 7832261 1906 55.-.G;132.G.0 0.876 0.807 ' , r., , 8530925 1880 75.-.G;82.-.A 0.891 0.434 15161180 1907 -29.A.G;77.-.A 0.875 0.216 8142901 1881 76.G.-;134.G.T 0.890 0.290 8545164 1908 75.-.G;82.AA.-G 0.875 0.569 8142383 1882 76.G.-;125.T.G 0.890 0.343 7830386 1909 55.-.G;86.-.0 0.875 0.744 2054253 1883 0.TT.--;2.A.G;87.-.T 0.890 0.872 6077749 1910 15.TC.-A;76.G.- 0.875 0.859 8001281 1884 71.T.0 0.888 0.608 8148008 1911 76.G.-;86.C.- 0.875 0.187 6366788 1885 17.-.A;86.C.- 0.888 0.797 2278635 1912 0.T.-;88.-.G 0.874 0.725 12123821 1886 2.A.-;76.G.-;131.A.0 0.887 0.303 1041817 1913 -17.C.A;75.-.0 0.873 0.246 1-0 n 15159066 1887 -29.A.G;74.T.- 0.886 0.228 2465231 1914 1.TA.--;3.C.A;82.AA.-T 0.873 0.830 10072842 1888 19.-.T;87.-.A 0.886 0.612 2266703 1915 0.T.-;90.-.G 0.872 0.862 cp n.) o 1979426 1889 0.T.C;80.A.- 0.886 0.576 6625678 1916 18.C.-;78.-.0 0.872 0.580 n.) o 10193667 1890 18.-.G;82.A.- 0.886 0.828 8136927 1917 76.G.-;86.-.0 0.872 0.493 c,.) o 1252039 1891 -15.T.G;76.-.G 0.885 0.316 8093375 1918 75.-.A;131.A.0 0.871 0.335 un =
un index SEQ ID muts lindexed index SEQ
ID muts lindexed MI 95% CI

NO NO

2454809 1919 1.TA.--;3.C.A;72.-.A 0.870 0.736 8519380 1946 76.GG.-T;129.C.A 0.860 0.207 n.) o 1980576 1920 0.T.C;76.GG.-T 0.870 0.466 8493521 1947 76.-.G;98.-.T 0.859 0.735 n.) o 2271158 1921 0.T.-;132.G.0 0.870 0.383 8128428 1948 75.-.C;128.T.G 0.858 0.241 .6.

oe 442251 1922 -27.C.A;75.-.0 0.870 0.273 1248006 1949 -15.T.G;88.G.- 0.857 0.217 wc'e 2350399 1923 0.T.-;18.-.G 0.869 0.556 5585921 1950 10.T.C;76.G.- 0.855 0.371 8498008 1924 78.A.G 0.869 0.356 6127219 1951 14.-.A;78.A.- 0.855 0.493 8080600 1925 74.-.G;86.-.0 0.868 0.560 3007558 1952 1.TA.--;90.-.G 0.854 0.711 3328595 1926 2.A.G;0.T.-;78.AG.-T 0.868 0.824 10555821 1953 15.-.T;80.AG.-T 0.854 0.843 8467079 1927 78.AG.-C 0.868 0.422 12747339 1954 0.-.T;78.A.T 0.854 0.745 6459918 1928 16.-.C;77.-.A 0.866 0.523 14344892 1955 -25.A.C;75.-.0 0.853 0.296 2265855 1929 0.T.-;88.GA.-C 0.865 0.721 10310038 1956 17.-.T;77.-.T 0.853 0.647 P
15161451 1930 -29.A.G;79.G.- 0.865 0.291 4303315 1957 4.T.-;76.G.T 0.852 0.664 ,..
, r., n.) 8565376 1931 75.CG.-T;133.A.0 0.865 0.308 14786751 1958 -29.A.C;55.-.G 0.851 0.737 ' .3 .6.
,..
un 2684676 1932 0.T.-;2.A.C;131.A.G 0.864 0.347 15059318 1959 -29.A.G;0.T.-;2.A.C;76.-.G 0.851 0.285 "
r., , 6461858 1933 16.-.C;86.-.A 0.864 0.611 15240190 1960 -29.A.G;2.A.- 0.851 0.500 ' , r., , 3011807 1934 1.TA.--;132.G.0 0.863 0.396 6468525 1961 16.-.C;91.A.-;93.A.G 0.849 0.652 1905700 1935 0.TTA.---;3.C.A;86.-.0 0.863 0.792 2826831 1962 0.T.-;2.A.C;15.-.T;75.-.G 0.849 0.523 8440297 1936 81.GAA.-TT 0.863 0.410 8212871 1963 86.-.C;133.A.0 0.848 0.669 8752800 1937 55.-.T;75.-.0 0.862 0.546 3318144 1964 2.A.G;0.T.-;82.AA.-T 0.848 0.742 12721020 1938 0.-.T;75.-.0 0.862 0.449 1246180 1965 -15.T.G;75.-.A 0.847 0.337 441780 1939 -27.C.A;75.-.A 0.861 0.300 1982591 1966 0.T.C;66.CT.-G 0.847 0.442 10070497 1940 19.-.T;76.G.-;78.A.0 0.861 0.561 15166880 1967 -29.A.G;81.GA.-T 0.847 0.253 1-0 n 8112403 1941 76.-.A;132.G.T 0.861 0.584 1904171 1968 0.TTA.---;3.C.A;74.-.G 0.846 0.783 1-1002534 1942 -17.C.A;2.A.C;0.T.- 0.861 0.227 14635061 1969 -29.A.C;0.T.- 0.846 0.382 cp n.) o 3324612 1943 0.T.-;2.A.G;78.A.0 0.861 0.737 8565091 1970 75.CG.-T;126.C.A 0.845 0.207 n.) o -a 5 3030912 1944 1.TA.--;78.A.-;80.A.- 0.861 0.838 2725821 1971 0.T.-;2.A.C;77.GA.--;80.A.T 0.845 0.837 c,.) o 10182195 1945 18.-.G;76.GG.-C 0.860 0.462 4259960 1972 4.T.-;130.T.G 0.844 0.800 un =
un index SEQ ID muts lindexed index SEQ
ID muts lindexed MI 95% CI
MI 95% CI
NO NO

3135495 1973 1.T.G;3.C.-;75.-.G 0.844 0.791 10528065 2000 15.-.T;79.GA.-C 0.831 0.713 n.) o 14345120 1974 -25.A.C;76.G.- 0.844 0.259 3261986 2001 0.T.-;2.A.G;74.T.G 0.830 0.736 n.) o 10071193 1975 19.-.T;81.G.- 0.844 0.779 8131593 2002 75.-.C;99.-.G 0.830 0.553 .6.

oe 6476304 1976 16, C;78.AG.-T 0.844 0.661 14255597 2003 -24.G.T;2.A.- 0.830 0.570 wc'e 15175052 1977 -29.A.G;55.-.T 0.844 0.629 14879001 2004 -29.A.C;15.-.T;75.-.G 0.829 0.805 8519203 1978 76.GG.-T;126.C.A 0.843 0.233 14918841 2005 -- -29.A.C;2.A.-;76.GG.-C -- 0.829 -- 0.732 8173991 1979 77.GA.-- 0.843 0.383 2290589 2006 0.T.-;79.GA.-T 0.829 0.726 12746208 1980 0.-.T;76.-.G 0.842 0.435 2951795 2007 1.TA.--;16.-.0 0.829 0.306 8133056 1981 75.-.C;87.-.T 0.842 0.419 9987799 2008 19.-.G;86.-.G 0.827 0.731 8526626 1982 76.-.T;131.A.0 0.841 0.223 15455726 2009 .. -30.C.G;78.A.- .. 0.827 .. 0.282 1252968 1983 -15.T.G;75.C.- 0.841 0.361 14812695 2010 -29.A.C;77.-.T 0.826 0.575 P
14646713 1984 -29.A.C;0.T.-;2.A.C;80. A.- 0.840 0.513 8202480 2011 87.-.A;131.A.0 0.825 0.570 ,..
, r., n.) 6304778 1985 16.-.A;77.-.A
0.840 0.462 8066107 2012 74.T.-;121.C.A 0.825 0.204 ' .3 .6.
,..
o 8479746 1986 78.A.-;120.C.A 0.838 0.293 14807234 2013 -29.A.C;86.-.G 0.824 0.174 "
r., , 12763666 1987 0.-.T;55.-.T 0.838 0.783 10085211 2014 19.-.T;80.A.- 0.824 0.633 ' , r., , 2684656 1988 0.T.-;2.A.C;131.A.C;133.A.0 0.838 0.207 8180233 2015 81.GA.-C 0.823 0.428 14800177 1989 -29.A.C;79.G.- 0.837 0.233 1044371 2016 -17.C.A;87.-.G 0.821 0.293 8128118 1990 75.-.C;124.T.G 0.837 0.256 10286908 2017 17.-.T;85.TC.-A 0.821 0.502 13797685 1991 -14.A.C;0.T.-;2.A.0 0.836 0.250 10250881 2018 18.C.T;75.-.G 0.820 0.593 4259801 1992 4.T.-;128.T.G 0.836 0.763 2463586 2019 1.TA.--;3.C.A;86.-.G 0.820 0.682 6612829 1993 18.C.-;76.G.- 0.833 0.708 6554412 2020 18.C.A;76.G.- 0.819 0.318 448172 1994 -27.C.A;73.A.- 0.833 0.216 8485725 2021 76.-.G;98.-.A 0.818 0.716 1-0 n 1246589 1995 -15.T.G;76.GG.-C 0.833 0.560 2271237 2022 0.T.-;131.A.0 0.817 0.352 1-3 14796144 1996 -29.A.C;73.-.A 0.832 0.441 2564816 2023 0.T.-;2.A.C;17.-.A 0.816 0.601 cp n.) o 6611642 1997 18.C.-;76.GG.-A 0.831 0.704 8357229 2024 87.-.G;120.C.A 0.816 0.329 n.) o -a 5 3040392 1998 1.TA.--;73.A.T 0.831 0.517 12747630 2025 0.-.T;76.G.-;78.A.T 0.816 0.796 c,.) o 1938331 1999 0.TT.--;2.A.C;79.G.- 0.831 0.783 9972115 2026 19.-.G;73.-.A 0.816 0.802 un =
un index SEQ ID muts lindexed index SEQ
ID muts lindexed MI 95% CI
MI 95% CI
NO NO

8212329 2027 86.-.C;121.C.A 0.815 0.514 4011043 2054 3.-.C;74.-.0 0.799 0.713 n.) o 14654311 2028 -29.A.C;1.TA.--;76.G.- 0.815 0.380 14920334 2055 -29.A.C;2.A.-;86.C.- 0.799 0.460 n.) o iz..1 1864798 2029 0.TT.--;73.AT.-G 0.814 0.762 13845318 2056 -14.A.C;76.GG.-A 0.799 0.188 .6.
--.1 oe 8117352 2030 76.GG.-C;119.C.A 0.813 0.433 3427589 2057 0.T.-;2.A.G;19.-.G 0.799 0.416 wc'e 8479512 2031 78.A.-;119.C.A 0.812 0.224 14806422 2058 -29.A.C;89.A.- 0.798 0.702 8133372 2032 75.-.C;82.A.- 0.812 0.357 15165304 2059 -29.A.G;87.-.T 0.797 0.463 10468894 2033 16.C.-;87.-.G 0.812 0.667 2125941 2060 0.TTA.---;3.C.G;89.A.- 0.797 0.791 8489702 2034 76.-.G;121.C.A 0.812 0.335 15168973 2061 -29.A.G;76.-.T 0.796 0.380 14919783 2035 -29.A.C;2.A.- 0.812 0.513 8538239 2062 75.-.G;131.AG.CC 0.796 0.429 8198335 2036 86.C.A 0.811 0.799 8528721 2063 76.GGA.-TT 0.796 0.447 8105698 2037 76.GG.-A;133.A.0 0.811 0.269 7834109 2064 55.-.G;86.-.G 0.794 0.596 P
13845556 2038 -14.A.C;76.GG.-C 0.809 0.491 8476335 2065 78.A.-;98.-.A 0.794 0.528 ,..
, r., n.) A 3011864 2039 1.T.--;132.G.T
0.809 0.352 8352802 2066 132.G.C;86.C.- 0.794 0.214 ' .3 .6.
,..
--.1 13222066 2040 2.A.G;-3.TAGT.----;76.GG.-A 0.809 0.597 10372832 2067 18.CA.-T;74.-.T 0.794 0.724 "
r., , 6471171 2041 16.-.C;82.A.- 0.808 0.510 8752727 2068 55.-.T;76.GG.-C 0.793 0.681 ' , r., , 8526572 2042 132.G.C;76.-.T 0.808 0.259 6460172 2069 16.-.C;77.-.0 0.792 0.474 8352868 2043 86.C.-;131.A.0 0.807 0.226 1245743 2070 -15.T.G;74.T.-0.792 0.347 10198068 2044 18.-.G;76.G.-;78.A.T 0.807 0.436 6469515 2071 16.-.C;88.-.T 0.792 0.645 8137025 2045 76.G.-;89.-.A 0.804 0.538 15241028 2072 -29.A.G;2.A.-;78.A.- 0.792 0.398 8629413 2046 66.CT.-G;88.G.- 0.803 0.320 2711056 2073 0.T.-;2.A.C;82.A.G 0.791 0.747 8105428 2047 76.GG.-A;126.C.A 0.803 0.240 1974296 2074 0.T.C;74.T.- 0.790 0.533 7947397 2048 66.CT.-A;87.-.G 0.802 0.362 8637058 2075 66.CT.-G;86.-.G 0.789 0.254 1-0 n 7835793 2049 55.-.G;76.GG.-T 0.802 0.735 8526611 2076 76.-.T;132.G.T 0.788 0.323 1-3 8140338 2050 76.G.-;116.T.G 0.802 0.306 8144153 2077 76.G.-;119.C.T 0.788 0.240 cp n.) o 12722736 2051 0.-.T;77.-.0 0.801 0.427 10566620 2078 15.-.T;73.A.0 0.788 0.613 n.) o 8757065 2052 55.-.T;86.C.- 0.801 0.559 8557775 2079 74.-.T;119.C.A 0.788 0.230 c,.) o 2398681 2053 1.-.A;75.-.A 0.801 0.641 8462867 2080 79.GA.-T 0.787 0.613 un =
un index SEQ ID muts lindexed index SEQ
ID muts lindexed MI 95% CI
MI 95% CI
NO NO

8549438 2081 75.C.- 0.787 0.425 447600 2288 -27.C.A;75.CG.-T 0.776 0.266 n.) o 8558414 2082 74.-.T;129.C.A 0.787 0.255 8143156 2289 76.G.-;126.C.T 0.776 0.346 n.) o iz..1 8105581 2083 76.GG.-A;129.C.A 0.787 0.259 1982252 2290 0.T.C;73.A.- 0.776 0.441 .6.
--.1 oe 2281703 2084 0.T.-;86.C.T 0.786 0.719 4255522 2291 4.T.-;115.T.G 0.776 0.764 wc'e 2400499 2085 1.-.A;76.G.-;78.A.0 0.785 0.482 8112417 2292 76.-.A;131.A.0 0.776 0.677 14920368 2086 -29.A.C;2.A.-;87.-.G 0.785 0.602 8083653 2293 74.-.G;121.C.A 0.775 0.434 8543253 2087 75.-.G;91.A.-;93.A.G 0.785 0.452 8539008 2294 75.-.G;120.C.T 0.775 0.361 8488707 2088 76, G;116.T.G 0.785 0.283 13750813 2295 -13.G.T;75.-.G 0.774 0.496 9979217 2089 19, G;86.-.0 0.783 0.612 8759144 2296 55.-.T;76.GG.-T 0.772 0.578 15162226 2090 -29.A.G;86.-.A 0.783 0.522 2684637 2297 0.T.-;2.A.C;131.AG.CC 0.771 0.251 12146137 2091 2.A.-;116.T.G 0.783 0.429 8032414 2298 72.-.0 0.771 0.299 P
5454231 2092 8.G.C;76.G.- 0.782 0.646 15165408 2299 -29.A.G;86.-.G 0.770 0.132 ,..
, r., n.) 2288382 2093 0.T.-;77.GA.--;83.A.T
0.781 0.648 8352728 2300 86.C.-;129.C.A 0.770 0.200 ' .3 .6.
,..
oe 8549424 2094 75.C.-;132.G.0 0.781 0.386 12191702 2301 2.A.-;78.A.-;131.A.0 0.769 0.497 "
r., , 6461529 2095 16.-.C;85.T.- 0.781 0.720 12751144 2302 0.-.T;74.-.T 0.769 0.417 ' , r., , 1090544 2096 2.A.- 0.781 0.530 2894079 2303 1, C;87.-.G 0.768 0.697 2282648 2097 0.T.-;84.-.T 0.779 0.667 8480622 2304 78.A.-;129.C.A 0.768 0.332 12149194 2098 2.A.-;131.A.G 0.779 0.440 8758901 2305 55.-.T;76.-.G 0.766 0.642 8142223 2099 76.G.-;124.T.G 0.779 0.273 8202090 2306 87.-.A;121.C.A 0.766 0.622 8199575 2100 86.CC.-A 0.779 0.611 2885067 2307 1.-.C;79.G.- 0.766 0.512 13854291 2281 -14.A.C;75.CG.-T 0.779 0.362 8202431 2308 87.-.A;132.G.0 0.765 0.537 8092813 2282 75.-.A;121.C.A 0.778 0.281 12191659 2309 2.A.-;78.A.-;132.G.0 0.765 0.596 1-n 8605540 2283 73.A.-;87.-.G 0.778 0.303 12149115 2310 2.A.-;133.A.0 0.764 0.439 1-3 68946 2284 0.T.-;2.A.0 0.778 0.250 2271200 2311 0.T.-;133.A.0 0.764 0.429 cp n.) o 12199248 2285 2.A.-;76.GG.-T;132.G.0 0.778 0.424 2252404 2312 0.T.-;74.T.G 0.763 0.476 n.) o 8093073 2286 126.C.A;75.-.A 0.778 0.370 8142993 2313 131.A.G;76.G.- 0.762 0.250 c,.) o 12149170 2287 2.A.-;131.A.0 0.776 0.527 446438 2314 -27.C.A;78. A.- 0.762 0.249 un =
un DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.

NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME

NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME:
NOTE POUR LE TOME / VOLUME NOTE:

Claims

WO 2020/247882 PCT/US2020/036505What is claimed is:

1. A variant of a reference CasX protein (CasX variant), wherein:
a. the CasX variant comprises at least one modification in the reference CasX
protein;
and b. the CasX variant exhibits at least one improved characteristic as compared to the reference CasX protein.

2. The CasX variant of claim 1, wherein the improved characteristic of the CasX variant is selected from the group consisting of: improved folding of the CasX variant;
improved binding affinity to a guide nucleic acid (gNA); improved binding affinity to a target DNA; improved ability to utilize a greater spectrum of one or more PAM sequences, including ATC, CTC, GTC, or TTC, in the editing of target DNA; improved unwinding of the target DNA;
increased editing activity; improved editing efficiency; improved editing specificity; increased nuclease activity;
increased target strand loading for double strand cleavage; decreased target strand loading for single strand nicking; decreased off-target cleavage; improved binding of non-target DNA
strand; improved protein stability; improved protein solubility; improved protein:gNA complex (RNP) stability; improved protein:gNA complex solubility; improved protein yield; improved protein expression; improved fusion characteristics or a combination thereof.

3. The Cas X variant of claim 1 or 2, wherein the at least one modification comprises:
a. at least one amino acid substitution in a domain of the CasX variant;
b. at least one amino acid deletion in a domain of the CasX variant;
c. at least one amino acid insertion in a domain of the CasX variant;
d. a substitution of all or a portion of a domain from a different CasX;
e. a deletion of all or a portion of a domain of the CasX variant; or f. any combination of (a)-(e).

4. The CasX variant of any one of claims 1-3, wherein the reference CasX
protein comprises the sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

5. The CasX variant of any one of claims 1-4, wherein the at least one modification is in a domain selected from:
a. a non-target strand binding (NTSB) domain;
b. a target strand loading (TSL) domain;
c. a helical I domain;

d. a helical II domain;
e. an oligonucleotide binding domain (OBD); or f. a RuvC DNA cleavage domain.

6. The CasX variant of claim 5, comprising at least one modification in the NTSB domain.

7. The CasX variant of claim 5, comprising at least one modification in the TSL domain.

8. The CasX variant of claim 7, wherein the at least one modification in the TSL domain comprises an amino acid substitution of one or more of amino acids Y857, S890, or S932 of SEQ ID NO: 2.

9. The CasX variant of claim 5, comprising at least one modification in the helical I
domain.

10. The CasX variant of claim 9, wherein the at least one modification in the helical I
domain comprises an amino acid substitution of one or more of amino acids S219, L249, E259, Q252, E292, L307, or D318 of SEQ ID NO: 2.

11. The CasX variant of any one of claims 5-10, comprising at least one modification in the helical II domain.

12. The CasX variant of claim 11, wherein the at least one modification in the helical II
domain comprises an amino acid substitution of one or more of amino acids D361, L379, E385, E386, D387, F399, L404, R458, C477, or D489 of SEQ ID NO: 2.

13. The CasX variant of claim 5, comprising at least one modification in the OBD domain.

14. The CasX variant of claim 13, wherein the at least one modification in the OBD
comprises an amino acid substitution of one or more of amino acids F536, E552, T620, or 1658 of SEQ ID NO: 2.

15. The CasX variant of claim 5, comprising at least one modification in the RuvC DNA
cleavage domain.

16. The CasX variant of claim 15, wherein the at least one modification in the RuvC DNA
cleavage domain comprises an amino acid substitution of one or more of amino acids K682, G695, A708, V711, D732, A739, D733, L742, V747, F755, M771, M779, W782, A788, G791, L792, P793, Y797, M799, Q804, S819, or Y857 or a deletion of amino acid P793 of SEQ ID
NO: 2.

17. The CasX variant of any one of claims 5-16, wherein the modification results in an increased ability to edit the target DNA.

18. The CasX variant of any one of the claims 1 to 17, wherein the CasX
variant is capable of forming a ribonuclear protein complex (RNP) with a guide nucleic acid (gNA).

19. The CasX variant of any one of claims 1 to 18, wherein the at least one modification comprises:
a. a substitution of 1 to 100 consecutive or non-consecutive amino acids in the CasX
variant;
b. a deletion of 1 to 100 consecutive or non-consecutive amino acids in the CasX
variant;
c. an insertion of 1 to 100 consecutive or non-consecutive amino acids in the CasX; or d. any combination of (a)-(c).

20. The CasX variant of claim 19, wherein the at least one modification comprises:
a. a substitution of 5-10 consecutive or non-consecutive amino acids in the CasX
variant;
b. a deletion of 1-5 consecutive or non-consecutive amino acids in the CasX
variant;
c. an insertion of 1-5 consecutive or non-consecutive amino acids in the CasX;
or d. any combination of (a)-(c).

21. The CasX variant of any one of claims 1 to 20, wherein the CasX variant comprises two or more modifications in one domain.

22. The CasX variant of any one claims 1 to 21, wherein the CasX variant comprises modifications in two or more domains.

23. The CasX variant of any one of claims 1-20, comprising at least one modification of a region of non-contiguous amino acid residues of the CasX variant that form a channel in which gNA:target DNA complexing with the CasX variant occurs.

24. The CasX variant of any one of claims 1-20, comprising at least one modification of a region of non-contiguous amino acid residues of the CasX variant that form an interface which binds with the gNA.

25. The CasX variant of any one of claims 1-20, comprising at least one modification of a region of non-contiguous amino acid residues of the CasX variant that form a channel which binds with the non-target strand DNA.

26. The CasX variant of any one of claims 1-20, comprising at least one modification of a region of non-contiguous amino acid residues of the CasX variant that form an interface which binds with the protospacer adjacent motif (PAM) of the target DNA.

27. The CasX variant of any one of claims 1-20, comprising at least one modification of a region of non-contiguous surface-exposed amino acid residues of the CasX
variant.

28. The CasX variant of any one of claims 1-20, comprising at least one modification of a region of non-contiguous amino acid residues that form a core through hydrophobic packing in a domain of the CasX variant.

29. The CasX variant of any one of claims 23-28, wherein the modification is one or more of a deletion, an insertion, or a substitution of one or more amino acids of the region.

30. The CasX variant of any one of claims 23-28, wherein between 2 to 15 amino acid residues of the region of the CasX variant are substituted with charged amino acids.

31. The CasX variant of any one of claims 23-28, wherein between 2 to 15 amino acid residues of a region of the CasX variant are substituted with polar amino acids.

32. The CasX variant of any one of claims 23-28, wherein between 2 to 15 amino acid residues of a region of the CasX variant are substituted with amino acids that stack with DNA or RNA bases.

33. The CasX variant of any one of claims 1-5, wherein the at least one modification compared to the reference CasX sequence of SEQ ID NO: 2 is selected from one or more of:
a. an amino acid substitution of L379R;
b. an amino acid substitution of A708K;
c. an amino acid substitution of T620P;
d. an amino acid substitution of E385P;
e. an amino acid substitution of Y857R;
f. an amino acid substitution of I658V;
g. an amino acid substitution of F399L;
h. an amino acid substitution of Q252K;
i. an amino acid substitution of L404K; and j. an amino acid deletion of P793.

34. The CasX variant of any one of claims 1-5, wherein the CasX variant has a sequence selected from the group consisting of the sequences of Tables 3, 8, 9, 10 and 12, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%, sequence identity thereto.

35. The CasX variant of any one of claims 1 to 5, comprising a sequence selected from the group consisting of SEQ ID NOS: 258-327, 3508-3520, and 4412-4415.

36. The CasX variant of any one of claims 1-5, further comprising a substitution of an NTSB
and/or a helical lb domain from a different CasX.

37. The CasX variant of claim 36, wherein the substituted NTSB and/or the helical lb domain is from the reference CasX of SEQ ID NO: 1.

38. The CasX variant of any one of claims 1 to 37, further comprising one or more nuclear localization signals (NLS).

39. The CasX variant of claim 38, wherein the one or more NLS are selected from the group of sequences consisting of PKKKRKV (SEQ ID NO: 352), KRPAATKKAGQAKKKK (SEQ
ID NO: 353), PAAKRVKLD (SEQ ID NO: 354), RQRRNELKRSP (SEQ ID NO: 355), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 356), RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 357), VSRKRPRP (SEQ ID NO: 358), PPKKARED (SEQ ID NO: 350, PQPKKKPL (SEQ ID NO:
360), SALIKKKKKMAP (SEQ ID NO: 361), DRLRR (SEQ ID NO: 362), PKQKKRK (SEQ
ID NO: 363), RKLKKKIKKL (SEQ ID NO: 364), REKKKFLKRR (SEQ ID NO: 365), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 366), RKCLQAGMNLEARKTKK (SEQ ID
NO: 367), PRPRKIPR (SEQ ID NO: 368), PPRKKRTVV (SEQ ID NO: 369), NLSKKKKRKREK (SEQ ID NO: 370), RRPSRPFRKP (SEQ ID NO: 371), KRPRSPSS (SEQ
ID NO: 372), KRGINDRNFWRGENERKTR (SEQ ID NO: 373), PRPPKMARYDN (SEQ ID
NO: 374), KRSFSKAF (SEQ ID NO: 375), KLKIKRPVK (SEQ ID NO: 376), PKKKRKVPPPPAAKRVKLD (SEQ ID NO: 377), PKTRRRPRRSQRKRPPT (SEQ ID NO:
378), SRRRKANPTKLSENAKKLAKEVEN (SEQ ID NO: 379), KTRRRPRRSQRKRPPT
(SEQ ID NO: 380), RRKKRRPRRKKRR (SEQ ID NO: 381), PKKKSRKPKKKSRK (SEQ ID
NO: 382), HKKKHPDASVNFSEFSK (SEQ ID NO: 383), QRPGPYDRPQRPGPYDRP (SEQ
ID NO: 384), LSPSLSPLLSPSLSPL (SEQ ID NO: 385), RGKGGKGLGKGGAKRHRK (SEQ
ID NO: 386), PKRGRGRPKRGRGR (SEQ ID NO: 387), and PKKKRKVPPPPKKKRKV
(SEQ ID NO: 389).

40. The CasX variant of claim 38, comprising a sequence of any one of SEQ
ID NOS: 3540-3549.

41. The CasX variant of claim 38 or claim 39, wherein the one or more NLS
are positioned at or near the C-terminus of the CasX protein.

42. The CasX variant of claim 38 or claim 39, wherein the one or more NLS
are positioned at or near at the N-terminus of the CasX protein.

43. The CasX variant of claim 38 or claim 39, comprising at least two NLS, wherein the at least two NLS are positioned at or near the N-terminus and at or near the C-terminus of the CasX
protein.

44. The CasX variant of any one of claims 2-43, wherein one or more of the improved characteristics of the CasX variant is at least about 1.1 to about 100-fold or more improved relative to the reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ
ID NO: 3.

45. The CasX variant of claim 2-43, wherein one or more of the improved characteristics of the CasX variant is at least about 1.1, at least about 2, at least about 10, at least about 100-fold or more improved relative to the reference CasX protein of SEQ ID NO: 1, SEQ ID
NO: 2, or SEQ
ID NO: 3.

46. The CasX variant of any one of claims 2-45, wherein the improved characteristic comprises editing efficiency, and the CasX variant comprises a 1.1 to 100-fold improvement in editing efficiency compared to the reference CasX protein of SEQ ID NO: 2.

47. The CasX variant of any one of claims 1 to 46, wherein the RNP
comprising the CasX
variant exhibits greater editing efficiency and/or binding of a target sequence in the target DNA
when any one of the PAM sequences TTC, ATC, GTC, or CTC is located 1 nucleotide 5' to the non-target strand of the protospacer having identity with the targeting sequence of the gNA in a cellular assay system compared to the editing efficiency and/or binding of an RNP comprising a reference CasX protein in a comparable assay system.

48. The CasX variant of claim 47, wherein the PAM sequence is TTC.

49. The CasX variant of claim 47, wherein the PAM sequence is ATC.

50. The CasX variant of claim 47, wherein the PAM sequence is CTC.

51. The CasX variant of claim 47, wherein the PAM sequence is GTC.

52. The CasX variant of any one of claims 47, wherein the improved editing efficiency and/or binding to the target DNA of the RNP comprising the CasX variant is at least about 1.1 to about 100-fold improved relative to the RNP comprising the reference CasX.

53. The CasX variant of any one of claims 1 to 52, wherein the CasX variant comprises between 400 and 2000 amino acids.

54. The CasX variant of any one of claims 1 to 53, wherein the CasX variant protein comprises a nuclease domain having nickase activity.

55. The CasX variant of any one of claims 1-53, wherein the CasX variant protein comprises a nuclease domain having double-stranded cleavage activity.

56. The CasX variant of any one of claims 1-53, wherein the CasX protein is a catalytically inactive CasX (dCasX) protein, and wherein the dCasX and the gNA retain the ability to bind to the target DNA.

57. The CasX variant of claim 56, wherein the dCasX comprises a mutation at residues:
a. D672, and/or E769, and/or D935 corresponding to the CasX protein of SEQ ID
NO:1; or b. D659, and/or E756, and/or D922 corresponding to the CasX protein of SEQ ID
NO:
2.

58. The CasX variant of claim 57, wherein the mutation is a substitution of alanine for the residue.

59. The CasX variant of any one of claims 1 to 58, wherein the CasX variant comprises a first domain from a first CasX protein and second domain from a second CasX
protein different from the first CasX protein.

60. The CasX variant of claim 59, wherein the first domain is selected from the group consisting of the NTSB, TSL, helical I, helical II, OBD, and RuvC domains.

61. The CasX variant of claim 59, wherein the second domain is selected from the group consisting of the NTSB, TSL, helical I, helical II, OBD, and RuvC domains.

62. The CasX variant of any one of claims 59 to 61, wherein the first and second domains are not the same domain.

63. The CasX variant of any one of claims 59 to 62, wherein the first domain comprises a portion of the sequence selected from the group consisting of amino acids 1-56, 57-100, 101-191, 192-332, 333-509, 510-660, 661-824, 825-934, and 935-986 of SEQ ID NO: 1 and the second domain comprises a portion of the sequence selected from the group consisting of amino acids 1-58, 59-102,103-192, 193-333, 334-501, 502-647, 648-812, 813-921, and 922-978 of SEQ ID NO: 2.

64. The CasX variant of any one of claims 1-63, wherein the CasX variant is selected from the group consisting of CasX variants SEQ ID NO: 328, SEQ ID NO: 3540, SEQ ID
NO: 4413, SEQ ID NO: 4414, SEQ ID NO: 4415,SEQ ID NO: 329, SEQ ID NO: 3541,SEQ ID NO:
330, SEQ ID NO: 3542, SEQ ID NO: 331, SEQ ID NO: 3543, SEQ ID NO: 332, SEQ ID NO:
3544, SEQ ID NO: 333, SEQ ID NO: 3545, SEQ ID NO: 334, SEQ ID NO: 3546, SEQ ID NO:
335, SEQ ID NO: 3547, SEQ ID NO: 336 and SEQ ID NO: 3548.

65. The CasX variant of any one of claims 1 to 58, wherein the CasX variant comprises at least one chimeric domain comprising a first part from a first CasX protein and a second part from a second CasX protein different from the first CasX protein.

66. The CasX variant of claim 65, wherein the at least one chimeric domain is selected from the group consisting of the NTSB, TSL, helical I, helical II, OBD, and RuvC
domains.

67. The CasX variant of claim 65 or claim 66, wherein the first CasX
protein comprises a sequence of SEQ ID NO: 1 and the second CasX protein comprises a sequence of SEQ ID NO:
2.

68. The CasX variant of claim 66, wherein the at least one chimeric domain comprises a chimeric RuvC domain.

69. The CasX variant of claim 68, wherein the chimeric RuvC domain comprises amino acids 661 to 824 of SEQ ID NO: 1 and amino acids 922 to 978 of SEQ ID NO: 2.

70. The CasX variant of claim 68, wherein the chimeric RuvC domain comprises amino acids 648 to 812 of SEQ ID NO: 2 and amino acids 935 to 986 of SEQ ID NO: 1.

71. The CasX variant of any one of claims 1 to 5, comprising a sequence selected from the group consisting of SEQ ID NOS: 247-337, 3301-3493, 3498-3501, 3505-3520, 3540-3549 and 4412-4415.

72. The CasX variant of any one of claims 1 to 5, comprising a sequence selected from the group consisting of SEQ ID NOS: 247-337, 3498-3501, 3505-3520, 3540-3549 and 4412-4415.

73. The CasX variant of any one of claims 1 to 5, comprising a sequence selected from the group consisting of SEQ ID NOS: 3498-3501, 3505-3520, and 3540-3549.

74. The CasX variant of any one claims of 1 to 73, comprising a heterologous protein or domain thereof fused to the CasX.

75. The CasX variant of claim 74, wherein the heterologous protein or domain thereof is a base editor.

76. The CasX variant of claim 75, wherein the base editor is an adenosine deaminase, a cytosine deaminase or a guanine oxidase.

77. A variant of a reference guide nucleic acid scaffold (gNA variant) capable of binding a reference CasX protein or a CasX variant, wherein:

a. the gNA variant comprises at least one modification compared to the reference guide nucleic acid scaffold sequence; and b. the gNA variant exhibits one or more improved characteristics compared to the reference guide nucleic acid scaffold.

78. The gNA variant of claim 77, wherein the one or more improved characteristics is selected from the group consisting of: improved stability; improved solubility; improved transcription of the gNA; improved resistance to nuclease activity; increased folding rate of the gNA; decreased side product formation during folding; increased productive folding; improved binding affinity to a CasX protein; improved binding affinity to a target DNA
when complexed with the CasX protein; improved gene editing when complexed with the CasX
protein; improved specificity of editing when complexed with the CasX protein; and improved ability to utilize a greater spectrum of one or more PAM sequences, including ATC, CTC, GTC, or TTC, in the editing of target DNA when complexed with the CasX protein.

79. The gNA variant of claim 77 or 78, wherein the reference guide scaffold comprises a sequence selected from the group consisting of the sequences of SEQ ID NOS: 4-16.

80. The gNA variant of any one of claims 77 to 79, wherein the at least one modification comprises:
a. at least one nucleotide substitution in a region of the gNA variant;
b. at least one nucleotide deletion in a region of the gNA variant;
c. at least one nucleotide insertion in a region of the gNA variant;
d. a substitution of all or a portion of a region of the gNA variant;
e. a deletion of all or a portion of a region of the gNA variant; or f. any combination of (a)-(e).

81. The gNA variant of claim 80, wherein the region of the gNA variant is selected from the group consisting of extended stem loop, scaffold stem loop, triplex, and pseudoknot.

82. The gNA variant of claim 81, wherein the scaffold stem further comprises a bubble.

83. The gNA variant of claim 81 or claim 82, wherein the scaffold further comprises a triplex loop region.

84. The gNA variant of any one of claims 81-83, wherein the scaffold further comprises a 5' unstructured region.

85. The gNA variant of any one of claims 80 to 84, wherein the at least one modification comprises:

a. a substitution of 1 to 15 consecutive or non-consecutive nucleotides in the gNA
variant in one or more regions;
b. a deletion of 1 to 10 consecutive or non-consecutive nucleotides in the gNA
variant in one or more regions;
c. an insertion of 1 to 10 consecutive or non-consecutive nucleotides in the gNA variant in one or more regions;
d. a substitution of the scaffold stem loop or the extended stem loop with an RNA stem loop sequence from a heterologous RNA source with proximal 5' and 3' ends; or e. any combination of (a)-(d).

86. The gNA variant of any one of claims 77-85, comprising an extended stem loop region comprising at least 10, at least 100, at least 500, at least 1000, or at least 10,000 nucleotides.

87. The gNA variant of claim 85, wherein the heterologous RNA stem loop sequence increases the stability of the gNA.

88. The gNA variant of claim 87, wherein the heterologous RNA stem loop is capable of binding a protein, an RNA structure, a DNA sequence, or a small molecule.

89. The gNA variant of claim 87 or claim 88, wherein the heterologous RNA
stem loop sequence is selected from MS2, Q[3, Ul hairpin II, Uvsx, or PP7 stem loops.

90. The gNA variant of any one of claims 85-89, wherein the at least one modification compared to the reference guide scaffold of SEQ ID NO: 5 is selected from one or more of:
a. a C18G substitution in the triplex loop;
b. a G55 insertion in the stem bubble;
c. a Ul deletion;
d. a modification of the extended stem loop wherein i. a 6 nt loop and 13 loop-proximal base pairs are replaced by a Uvsx hairpin;
and ii. a deletion of A99 and a substitution of G64U that results in a loop-distal base that is fully base-paired.

91. The gNA variant of any one of claims 77-90, wherein the gNA variant comprises two or more modifications in one region.

92. The gNA variant of any one of claims 77-91, wherein the gNA variant comprises modifications in two or more regions.

93. The gNA variant of any one of claims 77-92, wherein the gNA variant further comprises a targeting sequence wherein the targeting sequence is complementary to the target DNA
sequence.

94. The gNA variant of claim 93, wherein the targeting sequence has 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides.

95. The gNA variant of any one of claims 93 or claim 94, wherein the targeting sequence has 20 nucleotides.

96. The gNA variant of any one of claims 93-95, wherein the gNA is a single-guide gNA
comprising the scaffold sequence linked to the targeting sequence.

97. The gNA variant of any one of claims 77 to 96, wherein the one or more of the improved characteristics of the CasX variant is at least about 1.1 to about 100-fold or more improved relative to the reference gNA of SEQ ID NO: 4 or SEQ ID NO: 5.

98. The gNA variant of any one of claims 77 to 96, wherein one or more of the improved characteristics of the gNA variant is at least about 1.1, at least about 2, at least about 10, or at least about 100-fold or more improved relative to the reference gNA of SEQ ID
NO: 4 or SEQ
ID NO: 5.

99. The gNA variant of any one of claims 77-98, comprising a scaffold region having at least 60% sequence identity to SEQ ID NO: 4 or SEQ ID NO: 5 exclusive of the extended stem region.

100. The gNA variant of any one of claims 77-98, comprising a scaffold stem loop having at least 60% sequence identity to SEQ ID NO: 14.

101. The gNA variant of claim 100, comprising a scaffold stem loop sequence of CCAGCGACUAUGUCGUAGUGG (SEQ ID NO: 245).

102. The gNA variant of any one of claims 77-101, wherein the scaffold of the gNA variant sequence has at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70%, at least 80%, at least 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the sequence of SEQ ID NO: 4 or SEQ ID NO: 5.

103. The gNA variant of any one of claims 77-101, wherein the scaffold of the gNA variant sequence comprises a sequence selected from the group of sequences of SEQ ID
NOS: 2101-2280, or having at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity thereto.

104. The gNA variant of claim 103, wherein the scaffold of the gNA variant sequence consists of a sequence selected from the group of sequences of SEQ ID NOS: 2101-2280.

105. The gNA variant of any one of claims 77-104, further comprising one or more ribozymes.

106. The gNA variant of claim 105, wherein the one or more ribozymes are independently fused to a terminus of the gNA variant.

107. The gNA variant of claim 105 or claim 106, wherein at least one of the one or more ribozymes are an hepatitis delta virus (HDV) ribozyme, hammerhead ribozyme, pistol ribozyme, hatchet ribozyme, or tobacco ringspot virus (TRSV) ribozyme.

108. The gNA variant of any one of claims 77-107, further comprising a protein binding motif.

109. The gNA variant of any one of claims 77-108, further comprising a thermostable stem loop.

110. The gNA variant of any one of claims 77-109, wherein the gNA is chemically modified.

111. The gNA variant of any one of claims 77 to 110, wherein the gNA comprises a first region from a first gNA and a second region from a second gNA different from the first gNA.

112. The gNA variant of claim 111, wherein the first region is selected from the group consisting of a triplex region, a scaffold stem loop, and an extended stem loop.

113. The gNA variant of claim 111 or claim 112, wherein the second region is selected from the group consisting of a triplex region, a scaffold stem loop, and an extended stem loop.

114. The gNA variant of any one of claims 111 to 113, wherein the first and second regions are not the same region.

115. The gNA variant of any one of claims 111 to 113, wherein the first gNA
comprises a sequence of SEQ ID NO: 4 and the second gNA comprises a sequence of SEQ ID NO:
5.

116. The gNA variant of any one of claims 77 to 115, comprising at least one chimeric region comprising a first part from a first gNA and a second part from a second gNA.

117. The gNA variant of claim 116, wherein the at least one chimeric region is selected from the group consisting of a triplex region, a scaffold stem loop, and an extended stem loop.

118. The gNA variant of claim 77, comprising the sequence of any one of any one of SEQ ID
NOS: 2101-2280.

119. The gNA variant of claim 77, comprising the sequence of any one of SEQ ID
NOS:
2236, 2237, 2238, 2241, 2244, 2248, 2249, or 2259-2280.

120. A gene editing pair comprising a CasX protein and a first gNA.

121. The gene editing pair of claim 120, wherein the CasX and the gNA are capable of associating together in a ribonuclear protein complex (RNP).

122. The gene editing pair of claim 120, wherein the CasX and the gNA are associated together in a ribonuclear protein complex (RNP).

123. The gene editing pair of any one of claims 120-122, wherein the first gNA
comprises:
a. a gNA variant of any one of claims 93-119; or b. a reference guide nucleic acid of SEQ ID NOS: 4 or 5 and a targeting sequence wherein the targeting sequence is complementary to the target DNA.

124. The gene editing pair of any one of claims 120-123, wherein the CasX
comprises:
a. a CasX variant of any one of claims 1-76; or b. a reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

125. The gene editing pair of any one of claims 120 to 124, wherein the first gNA comprises:
a. a gNA variant of any one of claims 93-119; and b. a CasX variant of any one of claims 1-76.

126. The gene editing pair of claim 125, wherein the gene editing pair of the CasX variant and the gNA variant has one or more improved characteristics compared to a gene editing pair comprising a reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID
NO: 3 and a reference guide nucleic acid of SEQ ID NOS: 4 or 5.

127. The gene editing pair of claim 126, wherein the one or more improved characteristics comprises improved CasX:gNA (RNP) complex stability, improved binding affinity between the CasX and gNA, improved kinetics of RNP complex formation, higher percentage of cleavage-competent RNP, improved RNP binding affinity to a target DNA, ability to utilize an increased spectrum of PAM sequences, improved unwinding of the target DNA, increased editing activity, improved editing efficiency, improved editing specificity, increased nuclease activity, increased target strand loading for double strand cleavage, decreased target strand loading for single strand nicking, decreased off-target cleavage, improved binding of the non-target strand of DNA, or improved resistance to nuclease activity.

128. The gene editing pair of claim 126 or claim 127, wherein the at least one or more of the improved characteristics is at least about 1.1 to about 100-fold or more improved relative to a gene editing pair of the reference CasX protein and the reference guide nucleic acid.

129. The gene editing pair of claim 126 or 127, wherein one or more of the improved characteristics of the CasX variant is at least about 1.1, at least about 2, at least about 10, or at least about 100-fold or more improved relative to a gene editing pair of the reference CasX
protein and the reference guide nucleic acid.

130. The gene editing pair of claim 126 or claim 127, wherein the improved characteristic comprises a 4 to 9 fold increase in editing activity compared to a reference editing pair of SEQ
ID NO: 2 and SEQ ID NO: 5.

131. The gene editing pair of claim 130, comprising a CasX selected from any one of SEQ ID
NO: 270, SEQ ID NO: 292, SEQ ID NO: 311, SEQ ID NO: 333, SEQ ID NO: 336, SEQ
ID
NOS: 3498-3501, SEQ ID NOS: 3505-3520, and SEQ ID NOS: 3540-3549, and a gNA
selected from any one of SEQ ID NOS: 2104, 2106, or 2238.

132. A composition comprising the gene editing pair of any one of claims 120-131, further comprising:
a. a second gene editing pair comprising the CasX variant of any one of claims 1-76, or the reference CasX protein of any one of SEQ ID NOS: 1-3; and b. a second gNA variant of any one of claims 77-119 or a second reference guide nucleic acid, wherein the second gNA variant or the second reference guide nucleic acid has a targeting sequence complementary to a different or overlapping portion of the target DNA compared to the targeting sequence of the first gNA.

133. The gene editing pair of any one of claims 120-132, wherein the RNP of the CasX
variant and the gNA variant has a higher percentage of cleavage-competent RNP
compared to an RNP of a reference CasX protein and a reference guide nucleic acid.

134. The gene editing pair of any one of claims 120-133, wherein the RNP is capable of binding and cleaving a target DNA.

135. The gene editing pair of any one of claims 120-132, wherein the RNP is capable of binding a target DNA but is not capable of cleaving the target DNA.

136. The gene editing pair of any one of claims 120-132, wherein the RNP is capable of binding a target DNA and generating one or more single-stranded nicks in the target DNA.

137. A method of editing a target DNA, comprising contacting the target DNA
with a gene editing pair of any one of claims 120-136, wherein the contacting results in editing or modification of the target DNA.

138. The method of claim 137, comprising contacting the target DNA with a plurality of gNAs comprising targeting sequences complementary to different or overlapping regions of the target DNA.

139. The method of claim 137 or claim 138, wherein the contacting by the gene editing pair comprises binding the target DNA and results in introducing a mutation, an insertion, or a deletion in the target DNA.

140. The method of claim 137 or claim 138, wherein the contacting introduces one or more single-stranded breaks in the target DNA and wherein the editing comprises introducing a mutation, an insertion, or a deletion in the target DNA.

141. The method of claim 137 or claim 138, wherein the contacting comprises introducing one or more double-stranded breaks in the target DNA and wherein the editing comprises introducing a mutation, an insertion, or a deletion in the target DNA.

142. The method of claim 140 or claim 141, further comprising contacting the target DNA
with a nucleotide sequence of a donor template nucleic acid wherein the donor template comprises a nucleotide sequence having homology to the target DNA.

143. The method of claim 142, wherein the donor template comprises homologous arms on the 5' and 3' ends of the donor template.

144. The method of claim 142 or claim 143, wherein the donor template is inserted in the target DNA at the break site by homology-directed repair.

145. The method of claim 142 or claim 143, wherein the donor template is inserted in the target DNA at the break site by non-homologous end joining (NHEJ) or micro-homology end j oining (MIVIEJ).

146. The method of any one of claims 137-144, wherein editing occurs in vitro outside of a cell.

147. The method of any one of claims 137-144, wherein editing occurs in vitro inside of a cell.

148. The method of any one of claims 137-144, wherein editing occurs in vivo inside of a cell.

149. The method of claims 147 or claim 148, wherein the cell is a eukaryotic cell.

150. The method of claim 149, wherein the eukaryotic cell is selected from the group consisting of a plant cell, a fungal cell, a protist cell, a mammalian cell, a reptile cell, an insect cell, an avian cell, a fish cell, a parasite cell, an arthropod cell, a cell of an invertebrate, a cell of a vertebrate, a rodent cell, a mouse cell, a rat cell, a primate cell, and a non-human primate cell.

151. The method of claim 149, wherein the eukaryotic cell is a human cell.

152. The method of claim 151, wherein the cell is an embryonic stem cell, an induced pluripotent stem cell, a germ cell, a fibroblast, an oligodendrocyte, a glial cell, a hematopoietic stem cell, a neuron progenitor cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, a retinal cell, a cancer cell, a T-cell, a B-cell, an NK
cell, a fetal cardiomyocyte, a myofibroblast, a mesenchymal stem cell, an autotransplated expanded cardiomyocyte, an adipocyte, a totipotent cell, a pluripotent cell, a blood stem cell, a myoblast, an adult stem cell, a bone marrow cell, a mesenchymal cell, a parenchymal cell, an epithelial cell, an endothelial cell, a mesothelial cell, fibroblasts, osteoblasts, chondrocytes, exogenous cell, endogenous cell, stem cell, hematopoietic stem cell, bone-marrow derived progenitor cell, myocardial cell, skeletal cell, fetal cell, undifferentiated cell, multi-potent progenitor cell, unipotent progenitor cell, a monocyte, a cardiac myoblast, a skeletal myoblast, a macrophage, a capillary endothelial cell, a xenogenic cell, an allogenic cell, or a post-natal stem cell.

153. The method of claim 151 or 152, wherein the cell is in a subject.

154. The method of claim 153, wherein editing occurs in the subject having a mutation in an allele of a gene wherein the mutation causes a disease or disorder in the subject.

155. The method of claim 154, wherein the editing changes the mutation to a wild type allele of the gene.

156. The method of claim 154, wherein the editing knocks down or knocks out an allele of a gene causing a disease or disorder in the subject.

157. The method of claim 151, wherein editing occurs in vitro inside of the cell prior to introducing the cell into a subject.

158. The method of claim 157, wherein the cell is autologous or allogeneic.

159. The method of any one of claims 147-151, wherein greater editing of a target sequence in the target DNA is achieved in a cellular assay system comprising an RNP
comprising the CasX
variant when any one of the PAM sequences TTC, ATC, GTC, or CTC is located 1 nucleotide 5' to the non-target strand of the protospacer having identity with the targeting sequence of the gNA in a cellular assay system, compared to the editing efficiency of an RNP
comprising a reference CasX protein in a comparable assay system.

160. The method of any one of claims 149-159, wherein the method comprises contacting the eukaryotic cell with a vector encoding or comprising the CasX protein and the gNA, and optionally further comprising the donor template.

161. The method of claim 160, wherein the vector is an Adeno-Associated Viral (AAV) vector.

162. The method of claim 161, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-Rh74, or AAVRh10.

163. The method of claim 160, wherein the vector is a lentiviral vector.

164. The method of claim 160, wherein the vector is a non-viral particle.

165. The method of claim 160, wherein the vector is a virus-like particle (VLP).

166. The method of any one of claims 160-164, wherein the vector is administered to a subject in need using a therapeutically effective dose.

167. The method of claim 164, wherein the subject is selected from the group consisting of mouse, rat, pig, and non-human primate.

168. The method of claim 166, wherein the subject is a human.

169. The method of any one of claims 166-168, wherein the vector is administered at a dose of at least about 1 x 109 vector genomes (vg), at least about 1 x 1010 vg, at least about 1 x 1011 vg, at least about 1 x 1012 vg, at least about 1 x 1013 vg, at least about 1 x 1014 vg, at least about 1 x 1015 vg, or at least about 1 x 1016 vg.

170. The method of any one of claims 166-169, wherein the vector is administered by a route of administration selected from the group consisting of intraparenchymal, intravenous, intra-arterial, intracerebroventricular, intracisternal, intrathecal, intracranial, and intraperitoneal routes.

171. The method of claim 147, wherein the cell is a prokaryotic cell.

172. A cell comprising a target DNA edited by the gene editing pair or composition of any one of claims 120 to 136.

173. A cell edited by the method of any one of claims 137-165.

174. The cell of claim 172 or 173, wherein the cell is a prokaryotic cell.

175. The cell of claim 172 or 173, wherein the cell is a eukaryotic cell.

176. The cell of claim 175, wherein the eukaryotic cell is selected from the group consisting of a plant cell, a fungal cell, a protist cell, a mammalian cell, a reptile cell, an insect cell, an avian cell, a fish cell, a parasite cell, an arthropod cell, a cell of an invertebrate, a cell of a vertebrate, a rodent cell, a mouse cell, a rat cell, a primate cell, and a non-human primate.

177. The cell of claim 175, wherein the eukaryotic cell is a human cell.

178. A polynucleotide encoding the CasX variant of any one of claims 1-76.

179. A polynucleotide encoding the gNA variant of any one of claims 77-119.

180. A vector comprising the polynucleotide of claim 178 or claim 179.

181. A vector comprising encoding the CasX variant of any one of claims 1-76 and the gNA
variant of any one of claims 77-119.

182. The vector of claim 180, wherein the vector is an Adeno-Associated Viral (AAV) vector.

183. The vector of claim 182, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV-Rh74, or AAVRh10.

184. The vector of claim 180, wherein the vector is a lentiviral vector.

185. The vector of claim 180, wherein the vector is a virus-like particle (VLP).

186. The vector of claim 180, wherein the vector is a non-viral particle.

187. A cell comprising the polynucleotide of claim 178, or the vector of any one of claims 180-186.

188. A composition, comprising the CasX variant of any one of claims 1-76.

189. The composition of claim 188, further comprising:
a. a gNA variant of any one of claims 77 to 119, or b. the reference guide scaffold of SEQ ID NOS: 4 or 5 and a targeting sequence.

190. The composition of claim 188 or claim 189, wherein the CasX protein and the gNA are associated together in a ribonuclear protein complex (RNP).

191. The composition of any one of claims 188-190, further comprising a donor template nucleic acid wherein the donor template comprises a nucleotide sequence having homology to a target DNA.

192. The composition of any one of claims 188-191, further comprising a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

193. A composition, comprising a gNA variant of any one of claims 77 to 119.

194. The composition of claim 193, further comprising the CasX variant of any one of claims 1-76, or the CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

195. The composition of claim 194, wherein the CasX protein and the gNA are associated together in a ribonuclear protein complex (RNP).

196. The composition of any one of claims 193-195, further comprising a donor template nucleic acid wherein the donor template comprises a nucleotide sequence having homology to a target DNA.

197. The composition of any one of claims 193-196, further comprising a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

198. A composition, comprising the gene editing pair of any one of claims 120 to 136.

199. The composition of claim 198, further comprising a donor template nucleic acid wherein the donor template comprises a nucleotide sequence having homology to a target DNA.

200. The composition of claim 198 or claim 199, further comprising a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

201. A kit, comprising the CasX variant of any one of claims 1-76 and a container.

202. The kit of claim 201, further comprising:
a. a gNA variant of any one of claims 93 to 119, or b. the reference guide RNA of SEQ ID NOS: 4 or 5 and a targeting sequence.

203. The kit of claim 201 or claim 202, further comprising a donor template nucleic acid wherein the donor template comprises a nucleotide sequence having homology to a target sequence of a target DNA.

204. The kit of any one of claims 201-203, further comprising a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

205. A kit, comprising a gNA variant of any one of claims 77 to 119.

206. The kit of claim 205, further comprising the CasX variant of any one of claims 1-76, or the CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

207. The kit of claim 205 or claim 206, further comprising a donor template nucleic acid wherein the donor template comprises a nucleotide sequence having homology to a target sequence of a target DNA.

208. The kit of any one of claims 205-207, further comprising a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

209. A kit, comprising the gene editing pair or composition of any one of claims 120 to 136.

210. The kit of claim 209, further comprising a donor template nucleic acid wherein the donor template comprises a nucleotide sequence having homology to a target DNA.

211. The kit of claim 209 or claim 210, further comprising a buffer, a nuclease inhibitor, a protease inhibitor, a liposome, a therapeutic agent, a label, a label visualization reagent, or any combination of the foregoing.

212. A CasX variant comprising any one of the sequences listed in Table 3.

213. A gNA variant comprising any one of the sequences listed in Table 2.

214. The gNA variant of claim 213, further comprising a targeting sequence of at least 10 to 30 nucleotides complementary to a target DNA.

215. The gNA variant of claim 214, wherein the targeting sequence has 20 nucleotides.

216. The gNA variant of claim 214, wherein the targeting sequence has 19 nucleotides.

217. The gNA variant of claim 214, wherein the targeting sequence has 18 nucleotides.

218. The gNA variant of claim 214, wherein the targeting sequence has 17 nucleotides.

219. A CasX variant comprising substitutions L379R and A708K and a deletion of P793 of SEQ ID NO: 2.

220. A gNA variant comprising a sequence of ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG
UGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG (SEQ ID NO: 2238).

221. A gene editing pair, or composition, comprising the gene editing pair or composition of any one of claims 120 to 136, or a vector of any one of claims 180 to 186, for use as a medicament.

222. A gene editing pair, or composition comprising the gene editing pair, or composition, of any one of claims 120 to 136, or a vector of any one of claims 180 to 186, for use in a method of treatment, wherein the method comprises editing or modifying a target DNA;
optionally wherein the editing occurs in a subject having a mutation in an allele of a gene wherein the mutation causes a disease or disorder in the subject, preferably wherein the editing changes the mutation to a wild type allele of the gene or knocks down or knocks out an allele of a gene causing a disease or disorder in the subject.